Majalah Kedokteran Andalas

Majalah Kedokteran Andalas

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Microdialysis Study

INTRODUCTION

Ericaceous family plant produced some toxic substances, a diterpenoids, with perhydroazulene skeleton. Asebotoxin-III was found as a diterpenoid isolated from the leaves of asebi, Pieris japonica.(1) Death of enimal experiment from this toxin could occur by various ways such as: cardiac arrest, respiratory failure, hypersecretion, hypotension and convulsion.

Studies of ATX-III which introduced into cerebral ventricles of guinea pig produce hemorrhage into alveolar cavities of the lung.(2) The proposed that possibly massive symphatetic discharge will stimulate hypothalamus. However this hypothesis need to be tested to clarify the exact mechanism on the alteration of brain metabolism of autonomic nervous system.

The Harada N(3) concluded that on the mechanisms for the lung hemorrhage induced by ATX-III because of depolarization of some neurons in the hypothalamus guinea pig. He also proposed that the massive symphatetic discharge will produce severe pulmonary hypertension and hemorrhage.

Other researfhers, Maekawa et al.fund that ATX-III induced pulmonary hemorrhagic edema colud be prevented by inhibiting the depolarization of nerve cell membrane of symphatetic centre in the hypothalamus of the brain rat.(4)

Studies on the action of ATX-III on sodium channel were reported that ATX-III will produce a marked depolarization on the squid nerve do to a specific increased in resting sodium permeability.(5) Later, Seyama and Narahshi (6,14) found that grayanotoxin molecule caued a slow opening the sodium channel of squid nerve membranes. It was known that ATX-III was the most cardiotripic and toxic compound among the member of grayanoid toxin (7) suggested the grayanotoxin opened Na channels from inside the squid axonal membrane.

Takeya et al. also concluded thad GTX-I (grayanotoxin) as a Ericaceous family toxin increases the resting Na-permeability on the papillary muscles isolated from the guinea pig heart. Recently, Hotta et al. found that ATX-III increases net Na(+)-influx via Na+ Channel in guinea pig erythrocytes and heart respectively.(2,11-13)

Furthemore, Maekawa et al. reported that ATX-III-induced pulmonary hemorrhage edema could be prevented by inhibiting the depolarization of nerve cell membrane of symphatetic center in the hypothalamus of the brain rat.(4)

The alterations on brain metabolism could be followed by microdialysis method for measuring the extracellular fluid of neurotransmitters and its metabolites in animal experiment. Neurotransmitters and its metabolites were collected into the striatum or other brain regions. Nakano and Mizuno (in press 1995) suggested that striatal dopaminergic neuronal function reaches a maximum level at 2-3 months old rats.(15-22)

Dopamine (DA) is a brain neurotransmitter and precursor in the biosyinthesis of nor epinephrine (NE|) in post ganglionic Symphatetic fibres. Synthesis of NE within the terminals of adrenergic axons is associated with precursor DA in the axons. DA is metabolized with monoamine oxidase (MAO) and catechol-o-methyl transferase (COMT). DOPAC (3,4-dihydroxyphenylacetic acid) was derived from a newly synthesized pool or DA. Serotonim (5-hydroxytryptamine = 5 HT) was a serum vasoconstrictor substance. Serotonin-, NE- and DA-containing neurons have been shown to project to the mammalian spinal cord.(25)

Recently, Kobayashi and Amenta reported that neurotransmitter receptors in the pulmonary endothelium heve an importance role in the pathopyysiologicel changers in the pulmonary circulation.(23)

The aims of this study was to investigate the alterations of neurotransmitter receptors metabolism and its metabolites after administration of ATX-III into the brain ventricle by using microdialysis technique.

MATERIALS AND METHODS

Animals:

Adult male Sprague-dawley rats were obtained fram Japan SLC, Inc. (Hamamatsu, Japan). The animal were kept standard food and water adlibitum under a 12 hours light-dark cycle (lights on at 7:00 a.m). Experiments were started between 9:00-11:00 a.m. The animal experiments were conducted according to the guide-lines for animal experiment prescribed by the AichiMedicalUniversity.

Microdialysis:

Rats were anesthesized with sodium pentobarbital (Nembutal;50mg/kg body weidht and placed in a stereotactic apparatus. The skull was exposed, and small hole was drilled to allow implantation of a guide cannula into the striatum and nucleus accumbence. Stereotactic coordinates from bregma were A+1.0, L+2.5, V-4.0 (dura) for striatum and A+2.5, L+1.5, V-6.0 for nucleus accumbence consequtively according to the skull screw and dental cement. Microdialysis probe (BDP-I-8-03; molecular cut-off 50,000;- EICOM Co., Kyoato, Japan) were designed to extend 3 mm beyond the tip of the guide cannula.

HPLC analysis:

Brain microdialysis and HPLC (High Performance Liquid Chromatography) were perfomed with a fully automated system. Extracellular contents of neurotransmitters and its metabolites were detected with an electrochemical detector (ECD-100, EICOM Co., Kyoto). The dialysis probe was perfused with Ringer's solution (NaCL 148 mM, KCL 4.0 mM, CaC12 2.3 mM) at a flow rate of 2 ul/min by means of a high precision syringe pump. Perfusion was begun 20-24 hours after the operation. The HPLC column was MA-5ODS (4.6 x 150 mm), the mobile phase was 0.1 M sodium citrate-acetate buffer (pH = 3.5) containing 0.1 mM EDTA and 17% methanol.

Perfusion solution:

The dialysate were collected for 25 minutes intervals, and were applied directly onto the HPLC-ECD apparatus. The ATX-III containing solution was 10 ug per ml Ringer. ATX-III was purified from laboratory in NagoyaCityUniversity (Hotta et al. 1980). DA and its metabolites, DOPAC, HVA (homovanilic acid), NE, epinephrine, and HMPG (4-hydroxy-3-methoxy phenylethylene glycol) as well as 5 HT and its metabolite, 5HIAA (5-hydroxyindoleacetic acid) were determined in the perfusate solution by HPLC.

RESULTS

The perfusion of Ringer solution achieved a steady state conditioned at least after 50 minutes in brain striatum microdialysis technique. Then the concertration of DOPAC, HVA and 5 HIAA remained about 20% after 75 minutes perfusion with Ringer contaning ATX-III (Fig. 1).

The contents of DA and serotonin metabolites in the perfusate decreased after injected with ATX-III into striatum rat 5 months old (Fig. 2). DOPAC concentration reduced from 9.3 ng into 4.4 ng/25 ul while HVA level that from 3.9 ng into 1.3 ng/25 ul. It was found that 5HT metabolite as 5HIAA also reduced markedly from 1.5 ng into 0.2 ng/25 ul. But DA content in the perfusate was increased markedly from 1.1 pg into 9.9 pg/25 ul while 5 HT that from 2.6 pg into 7.2 pg/25 ul after 100 minutes injection with ATX-III (Fig.3)

The contents of ATX-III in nucleus accumbence area that DOPAC, HVA and 5HIAA decreased gradually into 60, 62 and 42% after 175 minutes injection (Fig.4). Content of DOPAC reduced from 0.5 ng into 0.34 ng/25 ul, HVA from 0.33 to 0.3 ng/25 ul and 5HIAA from 0.36 into 0.23 ng/25 ul after injection with ATX-III into nucleus accumbence (Fig.5). But content of DA and serotonin in the perfuse increased significantly after injection with Ringer containing ATX-III solution. DA concertration incrased from 1 pg into 1.5 pg/25 ul but 5HT level that almost three times that from 3.1 to 8.9 pg/25 ul (Fig. 6).

The effects of ATX-III on catabolisme of neurotransmitters showed that DA metabolites, DOPAC, HVA and MHPG gradually decreased as well as that serotonin metabolite, 5HIAA (n=3) Fig. 7 However release of DA, serotonin and NE increased after ATX-III injection into brain striatum rat in the same animal experiment (Fig. 8).

DISCUSSION

A typical data showed that after at least 50 minutes perfusion studies with a physiologic solution, the steady state conditioned was achieved in brain rat striatum. Then DA and serotonin catabolism decreased after direct injection of ATX-III into the brain ventricle. In different region of brain, nucleus accumbence as a autonomic nerve centre, also found a typical data with decreasing of dopamine and serotonin catabolism after ATX injection (n=1 or 2).

Dopamin and serotonin catabolism decreased since its metabolites markedly reduced after direct injection of ATX into striatum. However the NE level was also found to increase markedly almost in the same pattern with serotonin in the striatum ATX injection. Therefora a higher level of DA, serotonin and NE might be caused by ATX-III stimulation to the neurotransmitters release.

Effect of direct injection ATX-III into brain striatum would increased sympathomimetic activity of NE and serotonin.

Content of DA in perfuse ATX injection was found increased after 25 minute. In this perfusion study, DA level in striatum befofe ATX injection as a control animal are very low or undetectable. It is necessary to investigate the effects of ATX-III in high dopaminergic activity of brain area such as prefrontal cortex.

Dose of ATX-III during 25 minutes perfusion was calculated 0.5 ug. In the case of a higher ATX-III dose would be able to produce massive symphatetis discharge as their hypothesis by Takeya et al. (1981). Eventhough 2.0 ug ATX content after perfusion 100 min into brain striatum was injection with a slowly rate (2 ul/min).

Serotonin has a multiple actions, it have induced contraction of pulmonary arteries of bovine with a smooth muscle receptor (frenken and Kaumann 1984) Serotonin has been shown to elicit vasorelaxation by an endothelium-dependent receptor mechanism (Leff et al. 1987). Eventhough the lung was able to rapidly remove serotonin from circulation by a Na+_dependent transport mechanism (Strum and Junod 1987), the mechanism was in the rate limiting strep.

DA has a specific vasodilator effect, it has two sub-types of receptors in the periphery (Goldberg and Kohli 1983). The receptors was in the adventitial, the adventitial border and the intimal leyer of arteries (Amenta et al. 1990). The majority of DA receptors in the rabbit pulmonary artery are endothelial (Ricci et al. 1992). Recently Boundy et al. (1993) reported D3 dopamine receptor in rat brain. DA was able to elicit alpha adrenoceptor-mediated vasoconstriction in pulmonary circulatin an anesthersized dog (Shebuski et al. 1987).

Noradrenergic neurons of the rabbit pulmonary artery are endowed with presynaptic alpha-adrenoreceptor which determine the rate of impulse flow (Endo et al.1977). Other researchers (Greenberg et al. 1981) found that intralobar pulmonary srteries and veins in canine contract in response to nor-epinephrin. Rat pulmonary artery was reported content both alpha adrenoceptors mediating contraction and beta-1 and beta-2 adrenoceptors mediating relaxation (O'Donnell and Wanstall 1984).

In the conclusion of this study a high level of serotonin, DA and NE after direct injected ATX-III into brain ventricle would played an important role to produce a lung hemorrhage in animal experiment.

It was necessary a further investigation to study the effects of direct injection of ATX-III in other brain region. It was also to identify some unknown substances in the perfusat during microdialysis.

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Majalah Kedokteran Andalas No. 2. Vol.23. Juli- Desember 1999

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