Vasopressin mediates neuroprotection in mice by ...nopr. Department of Pharmacology, Christi an Medical

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  • Indian Journa l of Experimentai Biology Vol. 4 1, June 2003, pp. 574-580

    Vasopressin mediates neuroprotection in mice by stimulation of V I vasopressin receptors: Influence of PI-3 kinase and gap junction inhibitors

    Manoj G Tyagi* & K V Parthiban

    Department of Pharmacology, Chri sti an Medica l Co llege, Vellore 632 002, India

    Received 27 Decelllber 2002; revised 28 March 2003

    europroteeti ve effect of vasopressin analogues, argin ine Vasopressin (A VP) and lysine Vasopressin (L VP) was evaluated against MgCl, induced cerebral ischemia model. A VP significantly prevented (P < 0.01 ) MgCl2 (1M) induced cerebral ischemia as compared to lysine Vasopressin (LVP) which was less effecti ve (P < 0.05 ). Pretreatment wi th PI -3 kinase inhibitors, Wortmannin and L Y -294002 (50 flg /kg, ip) significantl y altenu:lted the protecti ve effects of vasopress in . A VP was al so effective in reducing the maxima l electroshock (MES ) induced convulsive time and th is protective effect was blocked by PI-3 kinase inhi bitors. On the ot her hand, pretreatment with gap junction intracellular commu nication (GJl C) blocker, mepiIenamic acid (30 mg/kg, ip) significantly potentiated the MgCI, induced cerebra l ischemia. This enhancement of cerebra l isc hemia was not reversed hy vasopressin analogue, L VP. The role of V I vasopressin receptor was evaluated by pretreating the anima ls with non-selec ti ve VI receptor antago ni st, des Gly-NH2' d (CH2)5 [D-Tyr2, Thr 41 OVT whi ch re- versed the effects of A VP suggesting a role for vasopressin V I receptors. Thi s study suggests that neurohypophyseal hor- mone, A VP is nl:uroprotecti ve against MgClz induced cerebra l ischemia and thi s effect is modulated by PI-3 kinase en zyme

    . inhibitors and protein kinase C inhibitors through possible influence on the cerebral vascular tone. This study suggests that ga p juncti ons have potential ro le in the induction of MgCl2 induced cerebral ischemia.

    Keywords: Arg ini ne vasopress in, Cerebral ischemia, Gap junction, Lysine vasopress in , Neuroprotecti on, PJ-3 kinase, Protein kinase C, Vasopress in receptors

    Cerebral ischem ia and haemorrhagic strokes resulting in ischemic ce ll death represent major cause of cere- brovasc ul ar di sorders. Various potenti a l treatment approaches have been developed to reduce the extent of tissue injury,approaches that have been derived from experimental models of cerebra l ischemia l.2 .

    Neuronal damage mechanisms depend on complex interactions between neurotranmitters, neuropeptides and infl ammatory molecules that modify survival and repair. A pathololgical co nsequence of cerebral ischemia is the hyperactivity of spec ific neuronal syn- apses and mechanisms targeting atten uation of excito- toxicity at the molecular receptor, and/or ion channel, protein and enzy me changes conti nue to be explored for therapeutic implications3. Drugs in tended for treatment of cerebral ischemi a might a lso be effective agai nst some for ms of epi lepsy. Some novel anticon- vulsant drugs emphasize this view as they also pre- vent cerebral ischemia due to constriction or occlu- sion of major cerebral arteri es4-6 .

    *Cnrrespondelll author: Tl:1 : (0416) - 262603 Fax : (0416) - 262788 E-ma il : lyagi243 @

    Arginine vasopress in (A VP) is an important hor- mone secreted by the posterior pitutary gland. A VP has also been shown to be a key regulator of physio- log ical homeostatic balance and acts on two types of vasopress in receptors V 1 and V2 in the brain. Vaso- pressin has been shown to have multifaceted action in the central nervous system affecting cogni- tion,behaviour and neuronal excitability7,8 . AVP has been fo und to induce depolarization of hippocampal neuro ns . A V P decreases the responsiveness of vaso- pressin neurons to acute changes in plasma osmo- lality. Data available o n A VP plasma level s in ischemic stroke are few and discordant5 . Several stud- ies suggest that A VP may have e ither pro- or anti- conv ul sant effect depending on the spec ific animal models of epilepsy , Vasopress in normally induces vasoco nstriction peripherally and also induces vaso- dilatation in some vascular beds by re leasing ni tric oxide and increasing cGMP levels9 .

    On the other hand, PI-3 kinases induce the phos- phorylation of inositol contain ing lipids. PI, PI-4P and PI (4 ,5) P2 at the hydroxy l position on the inositol ring. A VP has been shown to acti vate the Pl-3 kinase enzyme in some cell types 10- 12 The enzyme have been found to be expressed in the sy mpathetic and sensory


    neurons also in discrete sites of the brain. Neurotro- phic factors for e.g. BDNF utili ses the PI-3 kinase pathways fo r eliciting excitation in hippocampal neu- rons. Wortmannin , a potent PI-3 kinase enzyme in- hibitor has been shown to inhibit long term potenita- tion in the dentate gyrus of the rats 13. Recent studies suggest that PI-3 kinase mi ght also contribute in the cytokine induced neuroendocrine responses in some cell types.

    The importance of Mg2+ in homeostatic balance is well known. Mg2+ ion can cause inhibition of NMDA responscs in cul tured rat neurons and has been shown to pl ay all important role in neuronal depolarization. Mg2+ is an important ion for regul ati on of myogenic tone of cerebral blood vessels and it is be lieved to play an important role in the autoregulat ion of cere- bral blood flow. Dietary deficiency of magnesi um (Mg2+) as well as abnormalities in Mg2+ metabo li sm has been suggested to play importan t ro les in hyper- tensio n, stroke, atherosclerosis and diabeti c vascu lar disease I4 . 15.

    We utilised MgCh for induci ng cerebral ischemia in tbis study. MgCl2 induced reversible global cerebral ischemia in mice is an excellen t ill vivo model l (; .1 7 Recent studi es suggest an inten:ction of Mg2+ ion and PI-3 kinase enzyme in maintaining the cerebral vaSCLl-

    , ~ . \

    iar tone-). The purpose nf til!s study was to determ ine the ro le

    of signa! Ii ng molecules in the neuroprotecti ve action of vasopressin an d to characterize the vasopressin receptor SUbtype responsible for neuroprorect ion. This study was hithcrto carried out to el ucidate the possible llcwoprotective role of vasopressin against MgCb induced cerebral ischemia and max imal elect roshock induced convuls ions in mice. The influe nce of PI-3 kinase enzyme activi ty, gap junc tions and nerve grow th fac tor was also evaluated.

    Materials and Methods Drugs usedfor this study-- Norm al Sali ne (CMCH

    Pharnncy , Vel lore), MgCi2 (G laxo India Ltd, Indi a), Argi ni ne Vasopressi n, Lys ine Vasopress in (S hree Ganesh Pharmaceuticals, India, Sigma India ltd), L Y 294002, Wonma nn in, Staurosporine and Nerve Growth Factor (A lol1lone Labs, Is rael), Mephenam ic Acid (Parke-Dav is Ltd. India). DesGly-NI-12 , d (Cl-1;1) S [D-Tyr2, Thr41 OVT (Dr.Maurice Man ni ng , Ohio, USA) .

    Anilllal Corc- Thi s study was conducted on albino Swiss mice of either sex weighing between 25 and 3S g. The an imals were kept under standard

    laboratory conditions and given food and water ad libitum . A 12 hr dark : li ght cycle was also main- tained.

    Induclion of cerebral ischemia - Swiss albino mice were utili sed for inducti on of cerebral ischemi a. Global cerebral ischemia was induced by the in trave- nous inj ection (0.05 ml ) of 1M MgCb into the tail ve in 16.17 The indicati ve sy mptoms of cerebral ische- mia were gasping, sedation and loss of righting reflex . The total time duration of ischemic epi sode beginning with onset and recovery of ischemic episode W;'l:, noted.

    Maximal electro shock method-- Max imal Electro Shock seizures were induced in the animals usin g a

    I · d . b d I' 1 S 1'-) Th . I ~ tec lnlque escn e eard er ". e alllma s were pre- treated with the tes t drugs pri or to the start of exper i- ments. The animals were subjected tu electro shock (60 mA/O.2 sec) via transauri cul ar electrodes attached to a electroconclusive meter. After induction of sei - zures, durat ion of toni c con vu ls ion, clonic con vul sion and total time to regain righti ng reflex or mortali ty of the ani mals were noted and tabulated .

    Biostatiscal evalualion- The data are represented

  • 576 INDIAN J EXP SIOL, JUNE 2003

    pre-treatment of AVP and LVP (10 I-lg/ kg, ip) pro- duced significant neuroprotection against 1M MgCh (0.05 ml, iv) induced ischemia i.e. a reduction in ischemic time by 78% and 37% respectively (P E 140-

    u 120 E .,

    .t::. 100-u .~ c 80- Il> If)

    '" 60-e u .:: 40- ~


    0- III



    Fig. 3 - Effect of GJlC bl ocker, mephenam ic ac id (30 mg/kg, ip) on MgCl2 ( 1M, 0.05 ml , iv) induced cerebral ischemia in mi ce and in the presence of L YP (10 f1g/kg, ip). The results are expressed 3S mean±SE. **P < O.Ol.

    iii' 160 ",'0 c: c:

    o 0 140 .- " '" Q)

    ~~ 120 c: '" o E 100 ut= '0 c: " .- 80 u '" 60 " 0> ¥ ~ 40


    We utili sed mephenamic acid, a anthranilic acid analogue and GJIC inhibitor for evaluating our studies on gap junction intra cellular communication. Pre- treatment of mice with gap junction inhibitor, mephenamic acid (30 mg/kg, ip) potentiated the ac- tions of MgCl2 on cerebral ischemi a by increasing the ischemic time by almost 70% and thi s enhancement was significantly unaffected by L VP. The results are