In vitro andin vivo release of neurokinin A-like immunoreactivity from rat substantia nigra

Neuroscience Vol. 27, No. 2, pp. 521-536, 1988

0306-4522/88 $3.00 + 0.00

Printedin Great Britain

IN J’ITRO AND IN VW0 IMMUNOREACTIVITY

Pergamon Press plc 0 1988 IBRO

RELEASE OF NEUROKININ A-LIKE FROM RAT SUBSTANTIA NIGRA

F. J. DIEZ-GUERRA,* D. J. S. SIRINATHSINGHJI and P. C. EMSONY MRC Group, Department of Neuroendocrinology, AFRC Institute of Animal Physiology and Genetics Research, Babraham, Cambridge CB2 4AT, U.K.

Abstract-In uiuoand in oitro perfusion techniques have been used to study the release of neurokinin A-like immunoreactivity from the rat substantia nigra. Potassium depolarization and electrical field stimulation evoked calcium-dependent release from nigral slices. Potassium depolarization was also effective in t&o. Tetrodotoxin (1 PM) completely blocked electrically stimulated release but only diminished release in response to depolarizing potassium. Neurokinin A-like immunoreactivity release showed frequency dependence and a clear facilitation phenomenon between 5 and 25 Hz. High-performance liquid chromatography analysis of the immunoreactivity released in vitro revealed the presence of neurokinin A, neuropeptide K and neurokinin B, along with their sulphoxide forms. A marked depletion of neuropeptide K and neurokinin B content was observed when the tachykinin content of the nigral slices was examined before and after stimulation. However, the neurokinin A content of the slices was unchanged or even increased, suggesting an accelerated processing of neurokinin A precursors during the stimulation. The tachykinin peptides were degraded at different rates by substantia nigra homogenates; degradation was fastest for neuropeptide K and slowest for neurokinin A. The addition of a mixture of peptidases inhibitors (thiorphan, phosphoramidon, bestatin and captopril) substantially reduced the degradation of all three tachykinins, but did not completely block degradation. GABA-A receptor antagonists such as bicuculline and, particularly, picrotoxin potentiated the stimulated neurokinin A-like immunoreactivity release in uitro, but the GABA-agonist muscimol had no effect. Picrotoxin was even more potent in uivo. The results presented in this study demonstrate that neurokinin A, neuropeptide K and neurokinin B can be released by depolarizing stimuli from rat substantia nigra. Furthermore, the features exhibited by this release suggest that these peptides may have a neurotransmitter/neuromodulator role in the rat substantia nigra.

INTRODUCTION

The recent isolation and characterization of neurokinin A (NKA; also called neurokinin alpha24 substance Kr’ or neuromedin L33), neurokinin B (NKB; also called neurokinin beta24 or neuromedin K23) and the amino-terminally extended form of NKA named neuropeptide K (NPK)4’ has considerably enlarged the tachykinin family of peptides in mammals, which was until recently only represented by substance P (SP). Molecular cloning techniques have shown that alternative RNA splicing of one single gene (preprotachykinin-A (PPT-A) gene)

*Present address: Centro de Biologia Molecular, Facultad de Ciencias, Universidad Autonoma de Madrid, Cantoblanco, 28949 Madrid, Spain. tTo whom correspondence should be addressed. Abbreviations: DADLE, [D-Ala*-, D-Let?-]-enkephalin; DAGO, [D-Ala’-, Glyols-]-enkephalin; EDTA, ethylenediaminetetra-acetate; HEPES, n-2_hydroxyethylpiperzine-N-2-ethanesulphonic acid; HPLC, highperformance liquid chromatography; -LI, -like immunoreactivity; NKA, neurokinin A; NKB, neurokinin B; NPK, neuropeptide K; -PPT, preprotachykinin; RIA, radioimmunoassay; SP, substance P; TFA, trifluoroacetic acid; U50488H, trans-3,4-dichloro-N-methylN[2-( 1-pyrrolidinyl)cyclohexy]benzeneacetamide; VTA, ventral tegmental area.

is responsible for the generation of three different SP precursors, namely alpha-, beta- and gammapreprotachykinin-A (PPT-A), two of which, beta-PPT-A and Gamma-PPT-A, also contain the sequence of NKA.“,26,“.35 Several regional distribution and immunocytochemical studies1.22,3’,36.40,~ have shown that NKA, NKB and NPK are concentrated in areas known to be rich in SP such as the midbrain, basal ganglia and spinal cord; however, the relative amounts of the different tachykinins varied between regions, suggesting region-specific expression of the PPTgenes. It is now also established that SP-containing neurons which are distributed mainly in the dorsal striatum project to the substantia nigra and provide most of the tachykinin content of the latter region.‘7,20*2’Indeed, the substantia nigra and, particularly, the pars reticulata, contains the highest concentration of SP and NKA in the rat brain.‘,” Although the common cellular response to tachykinins is excitation, differences in the actions of SP and NKA have been shown in several brain regions. SP exerts a selective inhibitory effect in the superficial cortical layers, an action not shared by NKA.” On the other hand, NKA is more effective than SP at exciting nigral dopaminergic neuronsI and at least 10 times more potent at increasing locomotor activ-

ity in rats when infused into the ventral tegmental area (VTA).” VTA injections of SP activate DA metabolism in the prefrontal cortex, while VTA injections of NKA alter DA metabolism in the nucleus accumbens but not the cortical site.5 These differences observed in the responses to SP and NKA correlate well with the distribution of specific binding sites for SP and NKA/NKB recently reported2~‘,4.29.4” and are consistent with the suggestion that the different tachykinins may interact with the various tachykinin receptors to produce a variety of different responses in much the same way as the multiple opiate ligands interact with the various types of opiate receptorsz5 Recent autoradiographic studies indicate that binding sites for NKA are present in much greater density than SF binding sites in the ventral mesencephalon. 2.32,42Also, subcellular distribution studies of the tachykinin content in rat striatum and substan-

tia nigra have shown a localization of these peptides in synaptosomes and vesicle fractions consistent with their possible role as neurotransmitters or neuromoduIators.6 In the present study, we have used in uitro and in vivo perfusion techniques to demonstrate the release of neurokinin A from rat substantia nigra in response to physiological stimuli and studied its possible modulation by other peptides and neurotransmitters associated with the basal ganglia.

EXPERIMENTAL

PROCEDURES

Maferials

Synthetic peptides ~ne~oki~n A, neurokinin B, neuropeptide IQ were purchased from Peninsula Laboratories Ltd (St Helens, U.K.) Bovine serum albumin (BSA, RIA grade), EDTA HEPES, N-(alpha-rhamnopyranosyloxy-hydroxyphosphinyl)-L-leucyl+tryptophan (phosphoramidon), bestatin, mus&mol, (- )bicuculline methiodide, picrotoxin, naloxone, 3-hydroxy-tyramine (dopamine), a~tylcholine, L-glutamic acid and glycine were obtained from Sigma Chemical Co. (Poole, U.K.). [D-Ala2-, D-Leu’-]-enkephalin (DADLE), [D-Ala2-, Glyo15-j-enkephalin @AGO) and thiorphan w&e from C&bridge Research Biochemicals (Harston. U.K.). Trans-3.4dichloro-N-methyl-N12-ll-ovr_ . _. ~oli~nyl~ycloh~xyi}~n~~~~~de (U!%488H) was a generous gift of Dr J. Bicknell. Captopril (1-[(2S)-3mercapto-2-methyl-1-oxo-propyl]-t-proline) was kindly provided by E. R. Squibb SC Sons, Inc. (Princeton, NJ, U.S.A.). [“‘IlNeurokinin A (2-[“‘I]-iodo-histidyl-NKA) was purchased from Amersham International plc (Amersham, U.K.). Animais Adult rats of our own inbred Wistar-derived strain weighing approx. 250 g were used for the experiments. All rats were maintained in a controlled environment (14 h of light daily; lights on at 0700 h; temperature at 20-22°C) with water and food available ad lib~t~. In vitro release experiments Rats were killed by cervical dislocation and decapitation, their brains quickly removed and a block of tissue containing the substantia nigra cut and glued (cyanoacrylate adhesive, RS ~rn~nen~) to a metal block for slicing with a Vibratome. Five coronal sections (250 pm) of each rat brain containing the substatia nigra (both pars compacta and pars

rctlculah) were cut in cold Iso-osmotic’ ~aliil~ iiledlu~!l (125 mM NaCI. 5 mM KCI, 2 mM MgSO,. 2 rnv C.t(‘1. 25 mM HEPES, pH 7.4). Substantia nigra slices were dissected with a scapel blade and loaded into small plvsttc chambers (150 ~1 internal volume. three slices per chamber) which trapped the tissue between two pieces of nylon mesh and fitted into a special purpose-built perfusion system. ‘The chambers contained a pair of platinum foil electrodes (5mm apart). which were connected to a Gr;iss SS-XX stimulator via slim&is isolation and constant current umts. After 30min perfusion (0.f ml:‘min) with warm (37 C’i oxygenated Yamamoto’s HEPES medium (125 mM Nat‘l. 5mM KCI. 2mM CaCl,. 2mM MeSO,. 50 uM EDTANa:. 5 mM u-glucose. 25mM HEPES (pfi 7.4): 0.1% (w L1 bovine serum albumin, 10 HIM bestatin, iOpM caproprtl. t PM phosp~o~dmidon and I PM th~o~~n), collection of 5-min fractions started and progressed for a maximum of POmin. Stimulation conditions are detailed in each experiment. Depolarizing potassium solutions were prepared hy iso-osmotic substitution of sodium by potassium. The perfusates were collected in polypropylene tubes containmg 0.25 ml of 0.4 M HCl, frozen immediately on dry ice .md

freeze-dried before rad~oimmunoassay (RIA). Once the experiment was completed, the tissue remaining in the chambers was collected, homogenized in 1 ml of 0.1 M HCI (Teflon--glass), centrifuged for 5 min in an Eppendorf microfuge and the supernatant removed, frozen. freeze-dried and assayed for NKA. The reiease of NKA-like inlmunoreact~vity (-Ll) collected in each fraction was expressed as a percentage of the tot31 NKA-Ll present in the tissue at the onset of the fraction considered. The electrically (or potassium) evoked overflow (also called net release) was calculated as the difference between total outflow during 10 min after the onset of each stimulation period and the estimated basal eiBux underlying this period. The basal efflux was assumed to decline linearly from the 5-min fraction collected before stimulation to the fraction

15 min after the onset of stimulation.

Each in uitro release experiment consisted of at least two experimental chambers and two controls and at the end the values for the experimental and control chambers were averaged separately; it is these accumulated means that were used for evaluation of statistical significance. Student’s two-tailed l-test was used to determine Ihe significance of differences between means 4 SE M. In vivo release experimenrs Rats were anaesthetized with chloral hydrate (300 mg/kg body wt and supplemented with 90 mg/kg at l-h intervals) and placed in a Kopf stereotaxic instrument fitted with atraumatic ear-bars. Body temperature was monitored and maint~n~ at 37°C. The push-pull eannulae used in this study were of the concentric design and were constructed in this laboratory. Each consisted of an internal 30-gauge stainless steel tube which extended 1.0 mm beyond the 22-gauge thin-wall stainless steel external tube (Cooper’s Needleworks, Birmingham, U.K.). The push-pull cannula was implanted into the pars reticulata of the substantia nigra at the following co-ordinates: 3.0mm posterior to bregma, 2.5 mm lateral to bregma, and 8.7mm ventral (from dura). The co-ordinates of Pellegrino” were used but we employed the modifications of Wishaw*5 to maintain in each rat an angle of 8” 29’ above the horizontal as measured between the interaural line and the rostra1 edge of the upper incisor bar. Following cannula implantation, perfusion of the nigral site was immediately initiated at a rate of 50 pl/min with the same medium used for in vitro release. Perfusion was accomplished with two identically calibrated Gilson peristaltic pumps (Anachem Ltd, Luton, U.K.). Ten-minute samples were continuously collected on ice in polypropylene tubes containing 0.1 ml of 0.4 M HCI, frorrn on dry ice and freeze-dried before RIA.

529

Neurokinin A release from substantia nigra High -performance liquid chromatography Reverse phase high-performance liquid chromatography (HPLC) was used for separation of NKA, NPK and NKB prior to RIA. The HPLC system consisted of a Waters Associates Model 204 liquid chromatograph equipped with a Model UK6 injector, two Model 6000A pumps, a Model 660 programmer for gradient elution and a PBondapak Cl 8 column (30 x 0.39cm). After examination of the elution conditions with synthetic pcptides, a linear gradient of acetonitrile (2444%) in 0.1% (v/v) trifluoroacetic acid (TFA) with a flow rate of 1 ml/min over a period of 25 min was used routinely. Fractions (0.5ml) were collected and processed for RIA after freeze-drying. Radioimmunoassay The NKA antiserum (R-65) has been characterized before.’ Briefly, the anti-NKA serum had 16% cross-reactivity with NPK. 10% with NKB. 32% with eledoisin. 14% with kassinin and 0.05% with SP. Under the conditions used, half-maximal inhibition by NKA was observed at 19 fmol/tube and l-250 fmol/tube was the working range of the assay for NKA. The assay range was 20-600 fmol/tube for NPK and l~5OOOfmol/tube for NKB. Sodium phosphate buffer (0.05 M, pH 7.4) with 0.1% bovine serum albumin was used as routine assay buffer. Standards and samples were diluted in 0.4 ml assay buffer and 0.1 ml of the label ([lz51]NKA, about 5000 c.p.m.) and 0.1 ml of diluted antiserum (final dilution 1: 240,000) were added. After incubation for 40-48 h at 4”C, separation of bound and unbound radioligand was achieved by adding into each tube 0.25ml of a suspension containing 0.25% (w/v) gelatin, 0.32% (w/v) Dextran T-70 and 3.2% (w/v) activated charcoal in 0.05 M sodium phosphate (PH 7.4) at 4°C. The tubes were centrifuged immediately (lO,OOOg; 15min) and the supematants and pellets separated by decantation and counted in a gamma-spectrometer (55% counting efficiency). The same procedures were applied for measurement of NKB and NPK extent that standard curves were constructed using NKB or- NPK as appropriate. Peptide standards were checked by HPLC and amino acid analysis. Histology Following completion of the push-pull perfusion experiments the animals were given a lethal dose of chloral hydrate, the brain removed and fixed in phosphate-buffered formalin. Sections (5 pm) were then cut in the frontal plane on a freezing microtome and stained with Cresyl Violet to verify cannula placements (Fig. 6).

RESULTS

General characteristics Basal levels of NKA-LI release in vitro were initially high, 2&30 pg NKA-LI/fraction, probably reflecting tissue damage during dissection. However, these high levels fell rapidly and stabilized within 20 min at a basal level of l-5 pg NKA-LI/fraction, (< 0.1% of the tissue content). Fraction collection started 30min after initiation of perfusion in all subsequent experiments. Potassium depolarization or electrical field stimulation evoked an overflow of NKA-LI which was detected in the perfusate (Fig. 1). A second period of stimulation, either electrical or potassium, elicited a much reduced release of NKA-LI (3@-50% of that obtained after the first stimulation). Ca2+ removal from the medium and its iso-osmotic substitution by Mg2+ and

Hi-K* m

Hi-K’ m *** I

0

20

40

60

80

TIME (minutes)

Fig. 1. Effect of electrical field stimulation and potassium depolarization on NKA-LI release from perfused nigral slices. Fraction collection started 30 min after the onset of the perfusion. (A) Electrical stimulation (3 mitt, 25 Hz, 2 ms square biphasic pulses, 5OmA) or (B) potassium depolarization (3 min, 45 mM K+) was applied 15 and 50 min after initiating fraction collection. Results are expressed as percentage of NKA-LI tissue stores at the moment of release and are means f S.E.M. of at least three separate experiments. *P < 0.05, l*P < 0.01, ***p < 0.001, significantly different from basal release (no stimulation). Non-paired two-tailed r-test.

addition of 1 mM EGTA resulted in the total blockade of NKA-LI release in response to both electrical stimulation (2 min, 25 Hz, 20mA, 0.87 +0.06% mean f S.E.M., n = 3, for controls; and 0.00 + 0.00% mean + S.E.M., n = 3, for calcium removal) and potassium stimuli (potassium depolarization 2min, 45mM K+, 1.59+0.15%, meanf S.E.M., n = 3, for controls; and 0.00 fO.OO% mean + S.E.M., n = 3, for calcium removal). Addition of 1 FM tetrodotoxin completely prevented NKA-LI release evoked by 2 min of electrical stimulation (25 Hz, 20 mA) (0.91 f 0.13% for control and 0.30 f 0.00% of NKA-LI in the tissue for tetrodotoxin; mean f S.E.M.; n = 3) but only decreased the amount of NKA-LI released in response to 2 min of potassium depolarization (45 mM K+) (1.71 f 0.21% for control and 0.98 f 0.17% of NKA-LI in the tissue for tetrodotoxin; mean f S.E.M.; n = 3). Figure 5A shows the results obtained after in vivo perfusion of substantia nigra by means of push-pull

______-.. ______-__ A

*** T

lation periods at frequencies ranging ILom $ !(I \O Hr increasing amounts of immunoreactivity v+~‘rc‘d<,tected in the perfusate (Fig. 2). Stimulalion l’rcquencies lower than 5 Hz did not evoke a Ggnlticant increase over the basal levels. Analysis of the matcrlal released per unit pulse indicated a clear facilitation phenomenon between 5 and 25 Hz and no further increase at higher frequencies. of a depolarizing

potassium

Similarly. solution

applica[lcw

for 2 min CCC-

increasing amounts of NKA-Ll rclcasc ‘IS the potassium concentration was raised from 20 to 60mM (0.20 k 0.04% for 20 mM K in the IISU~ I'OI to 4.3 If: 0.17”;0 of NKA-Ll 60 mM Ki ; mean + S.E.M.; II = 3). Potassium concentrations lower than 20mM did not cvokc significant stimulated rcleasc. ited

High-perJormunce

I

10

1

20

I

I

30

FREQUENCY

40

I

50

(Hz)

Fig. 2. Frequency dependence. Perfused nigral slices were electrically stimulated (2 min, 2 ms square biphasic pulses, 20 mA) at 5, 10, 25 and 50 Hz, 45 min after initiation of the perfusion. Stimulated net release values were calculated and results are expressed as (A) percentage of NKA-LI tissue stores and (B) percentage of NKA-LI tissue stores per 600 electrical pulses. The results are means + S.E.M. of four separate experiments. *P < 0.005, **P < 0.01 ***P < 0.001, significantly different from basal release (no stimulation). Non-paired two-tailed r-test. cannulae. As can be seen, a 2-min pulse of highpotassium solution evoked a significant increase of NKA-LI release as compared with the basal outflow monitored during the preceding 60min. A second stimulation period applied 90 min after the onset of the first stimulus evoked a considerably lower amount of NKA-LI release. In an attempt to optimize electrically evoked NKA-LI release we tried different patterns of electrical stimulation. Stimulation in trains (4 s on, 6 s off during 5 min at 25 Hz, 20 mA) did not result in an enhanced release when compared to continuous stimulation (25 Hz, 20 mA during 2 min) (0.91 f 0.09% for trains and 0.95 f 0.06% of NKA-LI in the tissue for continuous stimulation; mean + S.E.M.; n = 3). Similarly, the use of nigral slices cut sagittally did not increase the efficiency of continuous stimulation at 25 HZ (20mA) for 2 min (0.86 _+0.07% for sagittal and 0.93 + 0.08% of NKA-LI in the tissue for coronal slices; mean f S.E.M.; n = 3). Frequency dependence When

nigral slices were exposed

to 2-min stimu-

liquid c~hromutogruph~

Reverse-phase of the perfusates, after stimulation of the nigral slices, revealed the presence in the perfusates of immunoreactive material eluting at the positions expected for the synthetic peptides NKA, NPK and NKB, consistent with stimulated release of all three peptides (Fig. 3). Small amounts of immunoreactivity were found corresponding to the sulphoxide forms NKA and NPK; unoxidized NKA was always the major immunoreactive form released. Basal release samples (not stimulated) did not contain sufficient NKA-LI to give detectable peaks after HPLC separation. Electrical and potassium stimulation, therefore, both evoked the significant release of all three peptides (NKA, NPK and NKB) although sometimes the amount of NKB was hardly detectable. Interestingly, when the tachykinin contents of the nigral tissue samples were compared after stimulation (Table 1) the tissue content of NKA was unchanged or even increased despite the release of NKA into the perfusates (Fig. 3 and Table 1). In contrast, the stimulated slice’s content of NPK was substantially reduced (Table I). This is most evident when the ratios of the tissue contents are compared. Thus the NKA : NPK ratio increases from 4.8 in unstimulated slices to around 9 after stimulation. However. the relative amount of each peptide detected in the perfusates was much closer to the ratios observed in the unstimulated tissue slices (Table 1). Degradation rues The various peptidase inhibitors used in the perfusion medium were chosen for their ability to inhibit peptidases implicated in neurokinin degradation.” In order to investigate their effectiveness as inhibitors of neurokinin degradation, homogenates of substantia nigra were incubated with and without the various inhibitors, and aliquots of the reaction mixture were taken at different times and their peptide content assayed (Fig. 4). Although big differences were not observed amongst the peptides, NPK showed the fastest degradation rate and NKA the slowest. The

531

Neurokinin A release from substantia nigra

ELUTION VOLUME (ml)

Fig. 3. HPLC analysis of NKA-LI released after stimulation. HPLC elution profiles of NKA-LI present in the perfusate after (A) ekctrical stimulation (5 min, 25 Hz, 2 ms square biphasic pulses, 50 mA) and (B) potassium depolarization {S tin, 45 mM K+). Perfusates from four chambers receiving identical stimulation were pooled, freeze-dried, resuspended in 0.1% TFA and injected into HPLC. The elution positions of the synthetic peptides and their sulphoxide forms are indicated by arrows. The dotted line indicates the percentage of acetonitrile in the mobile phase during the separation (flow rate: I ml/min; length of the run: 25 min).

Table 1. Effect of electrical stimulation and potassium depolarization on the tachykinin content of rat substantia niprra slices Stimulated net release (fmolj7 mg of tissue)

Tissue content (fmoi/7 mg of tissue) Before stimulation

Tachykinin Neurokinin A Neuropeptide

K

1028 f 28 (n = 9) 213 f 10 $ 7;)

Neurokinin 3 (h4;18;) NKA/NPK NKA/NKB

10:16

eWricaal stimulation 1156*56 (n = 6) 129 + 11*** (n = 6) 34 f 7 (; F.3) 34:oo

potassium depolarization

Electrical stimulation

1023 f 66 123 f 13’+ (n = 6) (n =5) 110 f 12*** 19 13** (n = 6) (n =6) 11*3** 31 t-5 (n = 3) ‘“9703) 6.47 33:OO 11.18

Potassium depolarization 159 * 19+* (n = 6)

25 f 4** (n = 6) 14 & 2** (?I =3) 6.36 11.36

Electrical stimulation (5 min, 25 Hz, 2 ms square biphasic pulses, 5 mA) and potassium depolarization (5 min, 45 mM Kf) were applied 45min after initiation of perfusion. Ten-minute perfusates from two chambers were pooled and freeze-dried. Tissue slices before and after stimulation were homogenized in I ml of cold 0.1 M HCI, centrifuged and the supernatants freeze-dried. Freeze-dried pellets were resuspended in 1ml of 0.1% TFA and analysed for tachykinin content. Results are expressed as fmol of the relevant tachykinin per 7 mg of tissue (equivalent to three nigral &es) and are means f S.E.M. of the indicated number of experiments. Tissue content before and after electrical or potassium depolarization was compared using a two-tailed Student’s t-test. *P <0*05.**Px0.01.***P< 0.001. All stimulated release samples were significantly increased above basal release values at l*P < 0.01. NSE. 1712-F

532

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occurrence had been demonstrated in the rat substantia nigra. The GABA-A receptor antagonists hicuculline and, more effectively, picrotoxin (which selectively binds to the associated chloride channel) potentiated stimulated NKA-LI release, whereas muscimol (A fully active GABA-agonist) and (-)baclofen (an agonist of GABA-B receptors) were devoid of effect. None of the opioid-related drugs tested significantly modified the stimulated NKA-LI release. Only the selective kappa-receptor agonist (IJ50488H) showed a tendency to reduce the levels 01 release, although it did not reach statistical significance. Finally, glycine addition significantly altered the amount of stimulated NKA-LI release, whereas dopamine, glutamate and acetylcholine were without effect. Of all drugs and neurotransmitters tested only picrotoxin (50 p M) affected the NKA-LI basal release levels (The addition of picrotoxin increased the basal levels from 2-4 pg NKA-LI~fra~t~~l~ to 8--10 pg NKA-Lijfraction.) In viva release of NKA-LI was also affected by the presence of picrotoxin (Fig. 5B). Thus, the addition of 50 FM picrotoxin prior to the second potassium stimulation resulted in a marked increase of both basal and stimulated NKA-LI release.

:““-r--:-:: *

;“::

011

’

0

’

20

’

’

40

’

’

1

DISCUSSION

60

TIME (minutes)

Fig. 4. Degradation of NKA-LI by substantia nigra homogenates. Substantia nigra (about 20mg of fresh tissue) dissected from rat brain was homogenized (Teflon-glass homogenizer) in 2 ml of Yamamoto’s HEPES medium with (+---•) and without (0-O) a mixture of peptidases inhibitors (1 pM thiorphan, 1 PM pho~hom~don, lOpM bestatin, 1OpM captopril). The homogenates were incubated at room temperature with constant stirring. Aliquots (0.3 mf) were taken at 0, 5, 15, 30 and 60 min and the su~matants injected onto the HPLC. NKA, NPK and NKB content was determined by RIA of the relevant HPLC fractions. Results are expressed as percentage of the initial content (0 min) and are means f S.E.M. of three separate experiments. Statistically significant, *P < 0.05, **P < 0.01, as compared with control homogenates (no added inhibitors). addition of the in~bitor mixture resuIted in a stower decline of the nigral peptide content, giving the

highest degree of protection to NKB. The t”’ values for the peptides in the absence of inhibition were NKA 50 min, NKB 4Omin, NKA 20min. In the presence of inhibitors the t”* values increased for all three peptides and were NKA and NKB > 60 min and NPK 60min. Nevertheless, the protection wnferred by the inhibitors was not complete for any of the tachykinins studied, probably due to the presence of peptidases in the homogenates which were not affected by the inhibitors used. Pharmacological study The results obtained are shown in Table 2. We concentrated on ne~ot~ns~tters or drugs known to interact with neurotransmitters or receptors whose

The results presented in this study demonstrate that endogenous NKA, NPK and NK3 in the rat substantia nigra can be released in response to depolarizing stimuli both in uiuo and in vitro. Furthermore, the fact that the electrically evoked release showed calcium dependence, tetrodotoxin sensitivity, freTable 2. Effect of drugs and neurotmnsmitters on electrically stimulated neurokinin-A-like immunoreactivity release from rat substantia nigra slices Stimulated net release (% NKA-LL in the tissue) ~-___l__l__-0.79 +- 0.09 (7) No drug 0.92 f 0.14 (6) Muscimol50 p M 1.07 f 0.07 (6)* (- )Bicuculline 50 p M 1.31 f 0.24(6)+* Picrotoxin 50 fi M 0.83 f 0.16 (5) [ - )Baclofen 50 p M Drug

DA00

5 PM

1.00*0.17(5)

DADLE S/JM U5@44gH 1OfiM Naloxone 5 p M Dopamine 10Of~M Glutamate 100 /r M Acetylcholine 100p M Glycine 1QOpM

1.10*0.19(5) 0.59 f 0.06 (6) 0.81 f 0.07 (6) 0.83 j, 0.11 (6) 0.96 f 0.14 (6) 0.85 f 0.07 (6) 1.22*0.13(5)*

Nigral slices (three per chamber) were electrically stimulated (2 min, 25 HZ, 2 ms square biphasic pulses, 2OmA) 6Omin after perfusion. DrQgs and n~otr~i~ were added 15 min before stimtdation. stimulated net release is calculated for two fractions and expressed as percentage of NKA-LI present in the tissue at the moment of release lmeanfS.E.M.: (n)i. Statistic&y different from controls (no drug) at ‘P < 0.05 or **p

<

0.01.

Neurokinin A release from substantia nigra A 50

40

30

20

/-

10

30

SO TIME

90

120

OF PERFUSION

150

180

210

(minutes)

Fig. 5. In uiuo release of NKA-LI from rat substantia nigra. Rats were anaesthetized and push-pull cannulae implanted in the substantia nigra. Perfusion was initiated immediately at a flow rate of 50 pl/min and IO-min fractions (0.5 ml) were collected. Pulses (20 min) of 45 mM potassium were given 60 and 150 min after the onset of perfusion. The second pulse was preceded by perfusion with (A) normal saline or (B) 50pM picrotoxin in saline. Results are expresed in pg of NKA-LI per fraction and are means & S.E.M. of six different experiments. Statistically significant, *P < 0.05, **P < 0.01, ***P < 0.001, as compared with basal levels (no stimulation). Non-paired twotailed t-test.

quency facilitation and modulation by at least GABA-related drugs would be consistent with the hypothesis that these peptides have a neurotransmitter/neuromodulator role in the rat subsmntia nigra. This observation of calcium dependency and, in the case of electrical stimulation, tetrodotoxin sensitivity makes it unlikely that release is due to non-specific tissue damage. Indeed, basal levels of NKA-LI released from the slices were uniformly low, consistent with the minimal tissue damage produced by use of the Vibratome. Previous studies have reported calcium-dependent tachykinin release from rat cerebral cortex and striatum slic&“ and capsaicin-evoked release from rat spinal cord. 39 In our study, both potassium and electrical stimulation were able to elicit calciumdependent NKA-LI release from nigral slices. In addition, potassium depolarization was also effective in vivo. When both kinds of stimulation are compared, the most relevant difference is their distinct sensitivity to tetrodotoxin. The fact that potassium-

533

evoked release was not fully blocked by tetrodotoxin suggests that a substantial portion of the stimulated release responds to direct depolarization of the nerve terminal and not to propagation of nerved impulses along the axons, as seems to be the case for electrically stimulated release. Electrically evoked release was only detectable above 5 Hz, a feature which seems to be characteristic of many neuropeptides in contrast to the lower frequencies required to detect release of amino acids or monoamines. It has been suggested that this phenomenon is due to rapid degradation of released peptides in situ; however, in a series of experiments using a variety of peptidase inhibitors, and prolonged periods of stimulation, Lee and Iversen2r were unable to detect somatostatin release below 15 mM potassium. Similarly, in recent experiments we also found (Bonanno and Emson, unpublished observations) that electrically evoked somatostatin release was not detectable below 5 Hz. Although we cannot rule out rapid degradation in situ, it seems more likely that peptide release may require higher cytosolic free calcium concentrations, as would be generated by high-frequency stimulation. Previous studies have shown that the pattern of presynaptic nerve activity affects the postsynaptic response by determining the type of neurotransmitter released.‘5,46 So it may be that different thresholds for

release will enable a neuron to release different proportions of co-existing peptides and “classical” transmitters from its terminals depending on the pattern of electrical activity. Of the three tachykinins recognized by our antiserum, NKA was the major form released. This was expected since NKA is, after SP, the most abundant tachykinin in the rat substantia nigra. Much lower amounts of the immunoreactive material released corresponded to NPK and NKB. Selective degradation could account for differences in the immunoreactivity recovered for each peptide. However, we did not find significant differences in the degradation rate of any of the three peptides in the presence of the peptidase inhibitors. On the other hand, we were surprised to see that the protection conferred by the inhibitors was not complete. Previous studies had demonstrated that 1 PM phosphoramidon and 100 PM bestatin almost completely blocked the degradation of NKA and NKB by striatal membranes.” Since we used a homogenate from substantia nigra and not a membrane fraction, it is probable that other soluble peptidases and proteases resistant to our inhibitor mixture were responsible for the degradation observed. The NPK and NKB contents in the nigral slices were substantially reduced by either potassium or electrical stimulation, whereas the slice content of NKA was unchanged or even in some cases increased. This striking observation can only be explained by the appearance after stimulation of a portion of NKA that was not detected before stimulation. Since neuropeptide synthesis is assumed to

F. J. DIE%-GUERRA et ul.

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Fig. 6. Representative

~-PM coronal section through the rat substantia nigra showing the push pull

perfusion site. CC, Central gray; Pr, substantia nigra. pars reticulata: large tIrrow. cannula site; small arrows, substantia nigra, pars compacta.

occur in the neuronal cell bodies and not in the nerve terminals, one obvious suggestion is that NPK, whose sequence contains NKA, suffers an accelerated processing somehow triggered during the stimulation. Indeed of the three tachykinins the tissue content of NPK was the most drastically reduced by stimulation. At the same time it is also likely that higher molecular weight forms containing the NKA sequence and not detected by our antiserum may contribute to the formation of new NKA. It has been recently shown that axons and terminals from SP-containing neurons contain SP precursors not detected by conventional SP antisera. These putative precursors can, however, be detected by antibodies raised against the SP-Gly sequence.*’ The possibility that the transient rise in cytosolic calcium during stimuiation is responsible for an accelerated processing of NKA precursors is supported by recent evidence suggesting a neuropeptide convertase activity as a major physiological function for proteinases belonging to the Ca-activated neutral proteinase family.‘* In our study, picrotoxin is the drug which most clearly affects NKA-LI release from rat substantia nigra. Previous experiments have shown that GABA (5~0 is able to inhibit potassium-evoked SP re-

lease from substantia nigra slices and that prcrotoxin and, bicuculline (although only partially) reverse that effect.‘” Our results show that muscimol did not inhibit stimulated NKA-LI release but picrotoxin and, to a lesser extent, bicuculline potentiated stimulated release. If SP and NKA are coexisting in the same synaptic vesicles and respond identically to stimulation, it is possible that addition of exogenous muscimol is not equivalent to the inhibitory effects of endogenously released GABA under our experimental conditions. This would explain why picrotoxin and bicuculline show a stimulatory effect on NKA-LI release in that they can remove the inhibitory effects of endogenous GABA. In neurophysiological studies, sitmulation of the caudate nucleus in vivo leads to a characteristic initial excitation of nigral neurons suggested to be due to tachykinin release, which is then foIlowed by a period of long lasting inhibition. 7*’ These observations suggest a double action for GABA released by caudate stimulation; a long-lasting synaptic inhibition of mgral neurons (a direct action) and termination of the excitatory action of tachykinins by inhibiting their release (an indirect action). (-)Bactofen, dopaminc and the opiate receptor ligands did not affect stimulated NKA-LI release, in agreement with the ohser-

Neurokinin A release from suhstantia nigra

that they did not affect SP release from nigral slicesi Only the kappa-selective agonist U50488H showed a tendency to inhibit NKA-LI release but this did not reach statistical significance. From the amino acids tested only glycine showed a modulatory effect on NKA-LI release whose physiological significance is difficult to explain at the present time. Recent studies show that rat substantia n&a contains only low densities of either type of the tachykinin binding sites described so far, those which preferentially bind to SP and to NKB.’ Nevertheless, vations

535

prior reports have claimed potent excitatory effects of NKA on nigral neurons” and high densities of NKA binding sites in the substantia nigra of guinea-pigs.38 The mechanisms of action and the physiological effects of the tachykinins in the substantia nigra still await further investigation. Acknowledgements-F. J. Diez-Guerra is grateful to the British Council for financial support. P. C. Emson Acknowledges the support of the Association to Combat Huntington’s Disease (Combat).

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