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. :. ~- , - nype~.:~trychiiiri,erg1c_, . ~tirtergic ~ah9 ItlOrphinergtC. ~C in Telation to .. . · · ~ _ · ,'· :.::-ncliropsycbiattj¢. disdlses _-·_: ·' ·

·. t~ -· . ~- .•• ~ ,1 ·.~ :-:--!, .... ~ - ~ .. ; .-_ :·. ; .... ·_·. . . ~ . . .

· ... -- '"f,.. RavikumM ·. · .. · : ·'

• • j - ' • .: ' : • • ' . • • • • .. • • \ . I " • • .,_ • • '

Department o(Neurology, Medical College Hospital, Trivftl1drum <i950 II , India ~ . . . .

and

· P ANn, K V D~tpa Deyi ~ f> ·A K~up* .'

Department of Bi~hemfstry, University of Kerala, Kariavattom 695017, Indi a

Received 20 December /999; revised 22 March 2000 / .

. Previous work from this laboratory had demonstrated- the presence of endogenous morphine, strychnine and nicotine in the mammalian brain and human serum samples. Morphine is synthesised fr9m tyrosine and .strychnine and nicotine from

· tryptophan . This study examines the Iole of strychnine, ·nicotine and ·mOrphine ·in neuropsychi atric disorders. The blood levels of tyrosine, tryptophan, strychnine, nicotine and morphine were .studied as also RBC membrane Na+-K+ A TPase activity. It was found that serum tyrosine levels were reduced and tryptophan levels elevated in all neuropsychiatric di sorders· stodied with a reduct_ion in RBC Na+-K+ A TPase activity. Nicotine was present 'in signifi<;ant amounts in serum of pati_ents with schizophrenia, ,CNS glioma anq syndroine X ;vith multiple lacpnar state. Mowhine was present in significant amounrs ~only in the serull) 9f pati00~s ""-~th _ multiple sc_ler<;~~is and ~DP. St£YI;hnine l'I(\S p~esent in signif~cant amol,!nts !n the

· serum Qf: patieius with epilepsy, ~~mSQn's: disease and MOP: 'fhe prese·nce of 11ic<;>tine ·and strychnine in significant . , : amq.unt~ equid be related to_-. ele~ate~ .. ~~fophan levels· ~~t:stiog th~ JYinbesis of ~h,es~ allqiloids from tryptophan.

··1 • < Morphtn(!'wa,s !lot ~etect6d itf Trio~~ 'qf:~,...,~rders. ow~~ ;lj:Jw}}'!Os ill_~ ~e!s not.ed in~~fD. Na:i--K+_·A l)'ase inhibition .· . · . no'ticed in most of the disortiers c6~ _t~(pelated' tD decreftsed' ~perp_olaAslng ,-m'ot'phill6rg1c transmjSsion and · increased

depolarising nicotinergic and ·strychin~ic transffii ssion. The role of morphine, stryclmine and nicotine in the pathogenesis of these di sorders in the setting of membrane Na+-K+ A TPase inhibition is dtscussed.

Morphine can be synthesized from intravenously injected salutariciine, thebaine and codeine and thebaine can be converted to morphine when incubated with microsomal preparation from liver, kidney and brain of rats1

• The higher affinity morphine receptor type is mu 1 and the lower affinity morphine-selective type is mu2. Recently Stefano has proposed the presence of a third mu 3 receptor to which morphine binds exclusively 1

•

It was suggested that as a class, the catecholaminergic neuron may give rise to dopamine, norepinephrine, epinephrine and morphine from tyrosine. Morphine immunoreactivity has been found in the hippocampus as well as in the mesocorticolimbic and mesostraital regions and morphine produces reinforcement associated with increased ventral tegmental area dopaminergic (VTA DA) neuron firing activation of mesolimbic pathway and an increase in nucleus accumbers (NAS) extracellular dopamine (DA) concentration 1

•

*Author fo r correspondence: Dr. P.A. Kurup, Gouri Sadan , T.C. 4/1525, North of Cliff House, Kattu Road, Kowdiar P.O., Trivandrum-695003, India.

Morphine plays a role in irnmunoregulation. Injection of vertebrate animals with morphine resulted in deficient macrophage function and alteration ofT­cell activity. Morphine tended to inhibit or reduce immunocyte actiVIty, i.e. chemotaxis, cellular velocity, phagocytosis and cellular responsiveness to peptidergic signal s1

• Opioid peptides are involved in regulation of the stress response and insulin induced hypoglycemia is associated with a 20% rise in CSF beta endorphin concentration 1• It has also been noticed that rat C-6 glioma cells contain opiate alkaloid binding receptor sites and it has been proposed to mediate an inhibitory effect of morphine of cell proliferation and metastasis 1

•

Strychnine is biosynthesized from tryptophan and a terpenoid C 10 unit. There are reports of strychnine binding sites in the brain. But so far no endogenous strychnine has been identified in mammalian brain and other tissues . Strychnine causes a blockade of central nervous system inhibition by selectively antagonizing the effect of glycine. Glycine is a inhibitory transmitter of 4-aminobutryrate receptor in

560 INDIAN J EXP BIOL, JUNE 2000

the central nervous system of vertebrates, particularly in the spinal cord2

. Ishimaru et at studied the strychnine insensitive glycine binding sites in cerebral cortex of chronic schizophrenic and suggested that an NMDA associated glycine binding site may be implicated in the pathophysiology of schizophrenia3

.

The pyridine ring of nicotine is derived biosynthetically from nicotinic acid, which is derived from tryptophan. The Pyrrolidine ring is derived from ornithine. Like acetylcholine, nicotine initially stimulates the autonomic ganglia, adrenal medulla and myoneural junction by rapidly depolari sing the cell bodies, but in large doses it produces persistent depolari sation and a blockade or paralysis of these structures4

. Newhouse et a! suggested involvement of nicotine receptors in neurodegenerative di sorders such as alzheimer's di sease (AD) and Parkinson's disease (PO) in which loss of nicotinic receptors has been described5

.

In thi s context it was considered pertinent to look for endogenous morphine, nicotine and strychnine in neuropsychiatric disorders. The disorders studied include - manic depressive psychosis, schizophrenia, primary generalised epilepsy, Parkinson's disease, multiple sclerosis and CNS glioma. The serum levels of tyrosine and tryptophan were also estimated as morphine is synthesised from the former and strychnine and nicotine from the latter.

Materials and Methods Clearance from the ethical committee of the

Medical College, Trivandrum was obtained for this study. Freshly diagnosed patients of primary generalized epilepsy, Parkinson's disease (PO), schizophrenia, manic depressive psychosis (Bipolar mood disorder), multiple sclerosis, CNS glioma and syndrome X with multiple lacunar state (diagnosed according to DSM III R criteria) were randomly selected from medicine and neurology wards of the Medical College Hospital, Trivandrum. Fasting blood was removed from each of the I 0 patients in each disease group (male, age 30-50 years) as well as from an equal number of age and sex matched healthy controls . The serum samples were pooled in each disease group and control and approximately 50 ml serum was used for the analysis of alkaloids. All patients and control subjects were NON-SMOKERS (passive or active). All blood samples were collected from the patients before starting treatment.

I . Pure nicotine, morphine and strychnine used in this study were obtained from Sigma Chemicals Co.,

USA. HPLC apparatus used was Waters (USA) and column (Supelcosil™ ABZ+ Plus) was obtained from Supelco (USA). The rotavapor used was from Buchi (Switzerland).

2. Extraction of human serum for alkaloids and their analysis

The following procedure which is a partial modification of that described by Carindale was used6

•7

• Concentrated HCI was added to the serum samples to a final concentration of I 0% and heated in a boil ing water bath for 30 min . It was then centrifuged at I ,000 g for 30 min. The precipitate was discarded and the pH of the supernatant was adjusted to 9.0 with NaOH solution (10%). The solution was saturated with NaCI and extracted with 5 volumes of I 0% n-butanol in chloroform. The organic phase was separated and concentrated to a small volume in a Buchi rotavapor. The concentrate was shaken with 0.01 N HCI thrice and the acid solution was collected. pH of this solution was then adjusted to 9.0 with dilute NaOH solution (O.IN) and the solution extracted with chloroform : methanol (70 : 30 v/v). The organic solvent layer was evaporated to dryness in N2 atmosphere and the residue dissolved in a small volume of chloroform : methanol (I: 1 v/v) . TLC of this solution was carried out over Silica gel G, using ethyl acetate:methanol :NH40H (80: 15:5) . Standards of pure nicotine, strychnine and morphine were also run . Nicotine had an Rf value of 0.93, morphine 0.58 and strychnine 0 .70. The standard spots were identified by exposure to iodine vapour and the spots corresponding to nicotine, morphine and strychnine in the extract were scraped out and eluted with chloroform : methanol (I : I v/v). The solvent was removed in vacuum and the residue di ssolved in 40 microL of acetonitrile: 25 mM potassium phosphate buffer p H.7 .0 (25:75). HPLC was carried out with 20 microL aliquots using the same of ·olvent system [Waters HPLC, Supelcosii™ ABZ+Plus (reversed phase) 5 Jlm, 15 em column, flow rate I ml/min., detection= UV -250nm]. These conditions were found to be optimum for the separation, detection and quantitation of these alkaloids in previous studies7

.

Nicotine had a retention time of 3.35 min, morphine 2.95 min and strychnine 8.33 min. Confirmation of the identify of the alkaloids in the serum extract was carried out by reHPLC with pure alkaloids. The HPLC fractions corresponding to individual alkaloids from the serum were collected and pooled. It was evaporated to dryness and the res idue digested with anhydrous methanol. The methanol solution was

RAVI KUMAR et al. : ALKALOIDAL NEUROTRANSMITIERS & 561

centrifuged and solvent evaporated from the solute in vacuum. It was then dissolved in the HPLC solvent and mixed with the solution of the pure alkaloid (purified by TLC) in each case and subjected to HPLC. Representative chromatograph of each of the alkaloids in tissue/serum extracts is provided in Fig. I.

Tryptophan was estimated by the method of David and William8 and tyrosine by the method of Wong et al9

. Dopamine was estimated by the method of Weii­Malherbe et al 10

• RBC membrane Na+-K+ ATPase activity was estimated by the method of Wallach and Kamath 11

•

0 .06

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Cll Cll c c :.c ·p

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2 4

NEUROPSYCHIATRIC DISORDERS

Results No nicotine could be detected in the serum of

control subjects. Patients of epilepsy, PD and MDP contained trace amounts of nicotine ( 1.25, 1.07 and 1.01 j..lg/100 ml serum respecti vely). On the other hand serum of patients with schizophrenia, CNS glioma and syndrome X with multiple lacunar state contained higher amounts of nicotine (5.287, 4.56 and 9.72 j..lg/dl respectively) .

No morphine could be esti mated in the serum of control subjects and the serum of patients with epilepsy, Parkinsons disease, Schizophrenia, CNS

{i)

Cll c

~ u > \!; (/)

L (b)

(c)

Cll c

\. I~ 6 8 10

Fig. Retention time (minutes) !lg/ml)

had a retention time of 3.35 min. and Strychnine (2 !lg/ml) had a retention time of 8.33 min ; (b) HPLC of TLC fractions of control serum; (c) HPLC ofTLC fractions of serum of patients with manic depressive psychosis.

.. ' • .,: ... ;·: :_·; > :· ·o ~ . ·- ~ ~~\':·~ - ~ ) .:-.<~-::~.~ ~ · · ~::: '·, :· ' .. _~564 · · ,- · ft-I£?1AN J.'E:XF~If)L;~ zooo: ·. ~ . ,~ ~t .._· . "'·> :-· .': ,.·, ·. · . •: . 1>: . :. :_ ~· __ .. ~ :~·:.· ;·, ·.· ,_:·-. · -·-~: ~ - J ~>· \; .-::: .. - ~. :~ ~: : . !~):-~.~)_~ ::.- :~ .---L~,:c~<, ~- ·- .~~)_ ~· :..-_:_.:_ : ~-.: ..

ghbma aoo· i~d~ome X i also showed .no peal( -~ . g,enerall~ . · epilq>§y: . : Parkfgsoo~s . disease ~ - · and · ... ~orrespcmding ~to. morph4w.. Serum of p~ti_ents wit~ . scbizophnrnia. :(Tabk~· t~ - - - _ · ·. · . · . . · . · ~DP ~ontai~d ' -9.56 ~dl while 1hat of muitip)e .'. ·- ·· RBC sildi~m pota~iilrti ATPase was found ·k> pe

.. - ~lera:sis ·had, 9:92-fJ.g/dL. :. ·. . . _:-. ·._·· · . '•:_ : ~~uce(!)r(piimari ,&Me~ljsed '• epi1~P-sy, J>ar~idSQn'$ . · . . ' ; ::~~ . strychili?e could_ ·be:~S~imated i~}h!'! : serum of.·. -: Aisea:~; ; ~ ·~ldp~~ :, ~~ho~$~--~~ :, QNS7: l ~ 8}~¥. · :. · .. ~~ subJ~~: Serujn' 6f~t~ents wfth ~pilep~y. 'Pft .. ~,; tl~iw~-~~~~~:_ ;x: ~j_~-il~l~pl~:~ .

. , · a~d: MDP. ;cq~.tai.~ t ~~~ -~·54 arid' . l,1~5LJ!g/4L_·~r~ • . ~- .st~t~~t~~}uo~ ~~(fabk~J.- · -~- ·Y ,~· : .. ,. · . . . stry.<;hnine:- tespep~~ve1y. · Serum :· of ·patients ~ : . ::_-:~: __ -~-. · -. -~~:;; __ .'. >·.' -~ .. .... ·:,- ~ - '-->. · ··-

- : schi~phrenia rmll~ple sderosis and · syfldrome ·X -:_ ~-- _. :: -. ~ -~ · ·· ·. -·, .._. ;.-' · · . · -·: ~ · _-. .. , : · _. . . : .'· ~- " . .' · : . . cont'~iried tra~es of _strychnine '(0.60, . 1.02 and ' 2.9_z':· . '!._, -~e it#r¥,'·l.n 'serii& ·;t(yptophllti'~ .docrea;e: in . -

llWdL respec.tively) .-·No strYchnine co~ict be detected · . ~rosine _in · ~ :serum of':patients of i:n.any. of tnese.· · .. · the S(:rUm of patients 'with CNS glioma. . disorders is -~ - stgnifi~aht _. observati6n it} tl)e ligllf of · Serum · fl:yptophan was found -to ·be elevated . ·in altcr-ed leveJs : of the alkaloidS. lt , is known that. primary generalised epilepsy, Parkinson's disease, · tryptopJian ·.is the .· precursor foi ' strychnine ~a~d multiple sclerosis, CNS glioma, schizophre~ia, MOP, · nicotine

2.4· The presence of strychnine and nicotine in

syndrome X with multiple lacunar state. Serum the serum _of patients of most disorders studied · may tyrosine levels were found to be decreased in primary be a reflection of their synthesis from tryptophan. The. _generalised epilepsy, · Parkinson's disease, absence of nicotine in MS and that of strychnine in schizophrenia, CNS glioma, syndrome X with CNS glioma in spite of increase tryptophan level may . multiple lacunar state. Dopamine levels were al so be a reflection of some block in their synthesis inspite found to be low in multiple sclerosis, CNS glioma, of increased availability of tryptophan. The absence Syndromf( X with multiple lacunar state, Primary of morphine in most ~isorders stu~ied is a reflection .

· · .- · · · ~~Jaw tyro~ and 9oparqine leyels:, which ~re- tl1f .. · _ Table I -Tyrosine and tryptophan catabolic patterns in · : preeurSors ~,.of·,..Qioq;h!Jle. · How_evey, . ~e · prese_uee . -6( ~ ·

neuropsychiatric disorders . . m~ine _. .in· ~- aiJd MS~ i~spite ·. oJ ·. dec::ret1sed . {Value~ are mean :±SD from 15 sa~ples} ' . tyrosine lei'~~~es"'fdrth6r.StU.d'y. r • :.. -~ .. ..- . · • . ~- -~ ,> .

• .. .'!,- t, • r ~ • • • .• • .. •

Groups

I Control 2 CNS glioma 3 MS 4 Epilepsy 5 PD 6 Schizophrenia 7 Syndrome-X 8 MDPManic

phase

Tryptophan (mg/dl)

!.II ±0.08 2.05 ±0.07" 1.99 ±0.07" 1.96±0.09 1.66±0.06" 2. 10±0.09" 1.80 ±0.06" 2.02±0.08"

Tyrosine (mg/dl)

1.14±0.09 1.01 ±0.07"

0.929 ±0.06" 0.883 ±0.05 0.901 ±0.06" 0.862 ± 0.05" 0.935 ± 0.06" 0.825 ± 0 .03"

Dop (ng/dl)

12.89 ±0.67 8.43 ± 0.44" 8.68 ± 0.52" 8.53 ±0.53" 8.45 ±0.44" 8.44±0.45" 8.92 ±0.5 1 a

8.0 1 ± 0.62"

Groups 2-8 has been compared with group I P values:"< 0.01 ; hbetween 0.01 and 0.05 .

Table 2 -Acti vi ty of RBC membrane Na+- K+ ATPase

Group

I Contro l 2 Epilepsy 3 PD 4 Schizophrenia 5 MOP 6 CNS glioma 7 MS 8 Syndrome-X

Membrane Na+-K+ ATPase activity (!-lg Pi/mg protein)

5.04 ±0.22 1 1.48 ±0. 139* 1.51 ±0.142* 1.24 ±0.130* 1.11 ±0.120* 1.94 ±0. 18*

0 .3 13 ±0.023' 1.05 ±0.12*

[Values are mean ± SD from 15 samples in each group] Groups 2-8 have been compared with group I . NS-not studied. *P<O.OI.

The i'nhibjtion . of membrane. N~+-K+ ATPase activity in most of the disorders studied is another significant observation. This inhibiti on can result from decreased hyperpolari sing morphinergic transmission and increased depolarising nicotinergic and strychninergic transmission2

.4 . It is known that inhibition of this enzyme leads to increase in intracellular calcium due to increase in sodium calcium exchange, increased entry of calcium via voltage gated calciu m channel and increased release of calcium from intracellular endoplasmic reticulum calcium stores 12

• The increased intracellular calcium by displacing magnesium from its binding sites leads to a decrease in functional availability of magnesium. Decrease in magnesium inhibits Na+-K+ ATPase further as ATP magnesium complex is the actual substrate for the reaction . Thus there is progressive inhibit ion of Na+-K+ ATPase 12

.

Strychnine displaces glycine from its binding site and inhibits glycinergic inhibitory transmi ssion in the brain . The glycine is free to bind to the strychnine insensi tive site of the NMDA receptor and promotes excitatory NMDA transmission with consequent increase in calcium load2

• Nicotine acts on the nicotinic cholinergic receptor which promotes membrane

.· .. ,., •. . • . .: 1

. l. ~ :._ ... _,:. :.~ . ~\ll K_~_c!~: ._Ai;._~DAL-J'ffiUR~~r:fTER.S & ; ~- ~ .. ~ l .. • .. -· ._ : _ • · • • ~ \'--> .. ... ,· · . :. ; . ~_;NEUROPSY.QIIA\.ffi.IC DIS(m.Df:RS . · - . , -. ' ·

. . \ ~

: ~6_3-

~·· '~ ~ .. •• \. - · ~ .... (". : .... • :., ; • • ~ • • 4 • • • '.(. • • • ~ .

de·po_l~~~~tio~ ~~d. i~crea~~-eJ1~~y- ()f .~~~U~ vja .thr . . mo~~~er~ic -~~n~~ssiqn i~ tb~ _ maQiC p~a~y' anq· , v~hage_ ~e~ .. c~n~r:n c~ls . -~p~ut~- , ~ depease· W tfle: ~e~~esstve phflSe '!\as ~n· -repbrted i~ .· h~perp~lansat~OJI. :O_f :Jbe .ne~~~_al _. ~~~a11~· ;. th~s . _ . t~e M~P. : · I~tbttton· <5f .N a+-K + AT~ c~h )l~So . resul~s 1~ Na+,.Kt ATPase sl?·"!~tOn: and ~uce'd > - I."~sult m ~ef~ttv~ neuronal membrane repolarisatip-n · open•!lt ' of the· vl)~~ 'g<t!e":.. _.e~ium · cfi~nfiel and· ' ll{ld a par.ox)'smal depol<tri~ation ·shift resulting ·.lri. decreas~ - ~ ih . intt-ac~hilat:_' cak!¥tti~- ,~ -. 'Fh.~ ·. ' both . - ~pileptogenesis 17 ~ . . ~- , . · · · · , .

increased nkotine an·d stry~hnine · and redUced · :increased intracellular Ca2+ activates the Ca2+

morphine can lead on to an .. i'ntmneuronal calcium' dependent ,c.alcipeurin signal transd~tion pathway . overloaded state and functicinal ·magnesium.deficiency . which can. produce T cell · activation and secretion ·of : owing to Na+-K+ ATPase inhibition. lnterleukin 3,4,5,6 and TNF alpha (Tumour necrosis

The changes disc~ssed above are with : respect to factor alpha)20

. This can explain the . immune the RBC membrane. It has been suggested that the activation in MS . TNF atpha binds to ·its receptor and changes in the RBC membrane may be reflective of in turn can activate the caspase cascade, especially the neuronal membrane c)J'anges_n_ If s-imj}ar changes take downstream. caspase-9 and produce apoptosis

21.

plat~ in ~he netiron·at membrane . also' (ftls can be Caspase~9 is. a'"! ICE protease which-converts IL-l beta studied only with isolated neuronal ~mtirane) then . precursor-to IL-l beta. IL-l beta produces apoptosis of the consequence of an inhibition of neuronal the neuron in Parkinson's disease and Alzheimers membrane Na+-K+ ATPase and the resultant increase disease and the oligodendrocyte, the myelin forming

· in neuronal calcium load and magnesium depletion cell in MS21

can be manifold. Na+-K+ ATPase inhibition can Increased intracellular Ca2+ can open the

produce neurotransmitter transport dysfunction, mitochondrial PT pore causing a collapse of the H+ apoptosis and mitochondrial dysfunction, protein gradient across the inner membrane and uncoupling of processing defects , immune activation and activation the respiratory chain

22. This also leads to volume

of oncogenes as discussed be low. dysregulation and rupture of the outer membrane of

The increased presynaptic neuronal Ca2+ can

produce cyclic AMP dependent phosphorylation of synapsins in the presynaptic neuron resulting in increased neurotransmitter release into the synaptic junction and ves icular recycling14

• Increased intracellular Ca2

+ in the post synaptic neuron can also activate the G-prote in coupled neurotransmi tter signal transduction system of monoamine neurotransmitters and also Ca2

+ dependent NMDA signal (glutamate receptor) transduction 15

• The plasma membrane neurotransmitter transporter (on the surface of the glial cell and presynaptic neuron) is coupled to a Na+ gradient which is disrupted by the inhibition of Na+­K+ ATPase, resulting in decreased clearance of neurotransmitter (monoamines and glutamate) by presynaptic and glial uptake at the end of synaptic transmission. By these mechani sms, inhibition of Na+-K+ ATPase can promote monoaminergic and glutamatergic transmiss ion. Increased g lutamatergic transmission resulting in exci totoxicity has been implicated in neuronal degeneration observed in Parkinson's disease 16

, primary generalized epilepsy' 7

and schizophrenia 18. Increased monoaminergic

transmi ssion particularly of dopamine in the mesolimbic system has been implicated in schizophrenia 18

• A biphasic response with increase in

mitochondria resulting in the release of AIF (apoptosis inducing factor) and cyto C (cytochrome C) to the cytoplasm. This results in activation of caspase 9 which produces cell death . Apoptosis has been implicated in neuronal degeneration22

• Increased neuronal apoptosis can produce defective synaptogenesis and synaptic connectivity contributing to functional disorders like schizophrenia and epileps/3

.

The magnesium deficiency related ATP synthase defect and increased calcium related opening of the mitrochondrial PT pore produce a mitochondri al dysfunction22

. This results in incomplete reduction of 0 2 and increased production of free radical, the superoxide ion. Mitochondrial dysfunction has been implicated in the pathogenesis of neuronal degeneration like Parkinson's disease24

. Increased intracellular Ca2

+ can also activate NOS (nitric oxide synthase) causing increased production of NO which combines with superoxide radical to form peroxynitrite ion promoting lipid peroxidation25

. Free radical damage has been implicated in oncogenesis and neuronal degeneration26

.

Intracellular magnesium deficiency also results in defective ubiquitin dependent proteolytic processing of glycoproteins and antigens as it requi res

564 INDIAN J EXP BIOL, JUNE 2000

magnesium for its function 27. The protein processing

defect can result in defective glycosylation of endogenous myelin glycoprotein antigens with consequent defective formation of MHC-antigen complex28

. The MHC linked peptide transporter is a P-glycoprotein which transports MHC-antigen complex to the antigen presenting cell surface and requires magnesium for its function29

. Intracellular Mg++ deficiency results in dysfunction of MHC linked peptide transport. Defective presentation of endogenous myelin glycoprotein antigen can explain the immune dysregulation in MS . A CD8 MHC class I restricted immune dysregulatory defect has been described in MS30

. Defective tumour antigen presentation to NK cell will lead to oncogenesis as cancer cell immunosurveillance becomes dysfunctional 31

. Defectively processed glycoproteins like membrane beta amyloid resist lysosomal digestion and accumulate producing neuronal degeneration 32

. Ubiquitin dependent proteolytic dysfunction has been reported in neuronal degeneration especially Parkinson's disease32

.

Defective glycoproteins and glycosaminoglycans of the neuronal membrane can produce defective synaptic connectivity producing functional disorders like epilepsy, MOP and schizophrenia33

. Defective glycosylation of proteins consequent to Na+-K+ ATPase inhibition can result in loss of contact inhibition and oncogenesis .

Increased intra cellular calcium activates phosphol ipase C beta which results in production of diacyl glycerol (DAG) which activates protein kinase C31

• The protein kinase C (PKC) activates the MAP kinase cascade resulting in cellular proliferation. The decreased intracellular Mg++ can produce dysfunction of GTPase activity of the alpha-subunit of G protein . The results in ras-oncogene activation , as more of the ras is bound to GTP rather than GOP. Phosphorylation mechanism are required for the activation of the tumours suppressor gene P53. The activation of P53 is impaired owing to intracellular magnesium deficiency producing a phosphorylation defect31

.

In syndrome-X there is a reduction tn

hyperpolarising morphinergic transmission and an increase in depolarising nicotinergic -and strychninergic transmission. This can lead to Na+-K+ ATPasc inhibition. The consequent increase in calcium within the cell especially the beta cell can displace magnesium from the binding site. Magnesium depletion within the beta cell can lead to

increased release of insulin from the beta cell. A cellular magnesium deficiency and increase in a calcium overloaded state can have the following consequences. (I) Increase in intracellular calcium can lead to immune activation and increased production of TNF alpha leading on to insulin resistance33

. (2) Intracellular cellular magnesium deficiency can lead to protein tyrosine kinase dysfunction, an insulin receptor defecr34

. (3) Increased intra cellular calcium can lead to increase G-protein coupled signal transduction of the contrainsulin hormones-glucagon, growth hormone and adrenaline. This leads to hyperglycemia. (4) Increased intra cellular calcium can open up the mitochondrial PT pore producing a mitochondrial dysfunction and uncoupling oxidative phosphorylation. Decreased intra cellular magnesium can inhibit ATP synthase producing decreased synthesis of A TP and a mitochondrial dysfunction. Decreased intracellular magnesium can lead to inhibition of glycolysis and citric acid cycle. Thus glucose utilization as a whole is decreased . (5) Intra cellular magnesium deficiency can produce decreased dolichol phosphate synthesis and N-linked glycosylation. Generation of ATP for synthesis of nucleoside diphosphate sugars for 0-linked glycosylation is also defective leading on to altered glycoproteins. Intra cellular magnesium deficiency can also upregulate GAG synthesis. Both these contribute to the microangiopathy and macroangiopathy of syndrome-X. (6) Increased intracellular calcium can increase the signal transduction of the G protein coupled platelet activating factor receptor and thrombin receptor producing thrombosis . Intracellular magnesium deficiency can also produce vasospasm described in syndrome-X.

Nicotine by its CNS stimulant action has been reported to promote epileptogensis4

. Strychnine by displacing glycine from its binding sites and decreasing inhibitory transmission in the brain promotes epileptogenesis2

. The imbal ance between hyperpolarising, morphinergic transmtss!On and depolarising, nicotinergic and strychninergic transmtss10n producing net Na+-K + ATPase inhibition can contribute to epileptogenesis. Inhibition of Na+-K+ATPase can lead to a paroxysmal depolarisation shift and epileptogenesis. No morphine was detected in epilepsy.

In schizophrenia glutamatergic excitotoxic mechanism has been described. Strychnine by blocking glycinergic transmission can contributes to

RAY! KUMAR et al.: ALKALOIDAL NEUROTRANSMITI'ERS & 565 NEUROPSYCHIATRIC DISORDERS

the decreased inhibitory transmission m schizophrenia2

. The glycine is free to bind to the NMDA receptor and promote NMDA transmission resulting in glutamatergic excitotoxicity. Nicotine by interacting with nicotinic receptors can facilitate the release of dopamine, promoting the dopaminergic transrrusswn in the brain4

. The dopaminergic hyperactivity in schizophrenia could be due to increased nicotine synthesis. No morphine was detected in schizophrenia possibly due to low tyrosine levels noted in the serum of these patients.

Increased levels of morphine, nicotine and strychnine has been detected in the serum of manic depressive psychosis patients. Morphine and nicotine have a biphasic effect on limbic dopaminergic and serotoninergic transmission with initial activation followed by significant inhibition later1

.4. This could contribute to the excessive monoaminergic transmission during the manic phase and reduced monoaminergic transmission during the depressive phase of bipolar mood di sorder. Strychnine may also act in a similar biphasic manner contributing to the bipolar mood disorder.

Morphine suppresses tumour growth and metastases while nicotinic cholinergic transmission promotes cellular proliferation 1.3

1• Morphine

deficiency and nicotinic excess could thus contribute to genesis of gliomas .

Strychnine was detected in Parkinsons disease. As mentioned earlier it can promote NMDA receptor ~ctivity, re~ulting in glutamatergi~ ex~itotoxi~i1t{ Important m neuronal degeneration m po-· . Increased nicotinic cholinergic transmiss ion can contribute to the tremor of Parkinson's di sease. No morphine could be detected in PD and the protective effect of morphine on intraneuronal calcium load is lost.

Nicotine may contribute to hypertension and hypertriglyceridemia observed in syndrome X by the vasospasm it produces and its reported effect on lipid metabolism respective!/. Strychnine was detected in syndrome X contributing to decreased Na+-K+ A TPase and altered calcium/magnesium ratios. Morphine deficiency was noticed in syndrome X. Intrathecal morphine administration produces hypoglycemia via spinal opiate and central alpha­adrenergic receptors' . Morphine also stimul ates insulin release from the beta cells 1

• Thus morphine deficiency can contribute to the hyperglycemia of syndrome X.

The detection of increased levels of morphine in multiple sclerosis is significant. Morphine has got a immunoregulatory function in the brain and has been found to inhibit the expression of antigenic markers in T helper and T suppressors cells 1• Morphine may contribute to this CD8 MHC class 1 restricted T-cell defect described in multiple sclerosis. Serum of patients with MS showed strychnine which can produce an increase in intraneuronal calcium load producing oligodendrocyte apoptosis and immune activation. No nicotine could be detected in MS.

In this context it is pertinent to note the interrelationship between these diseases documented in literature35

. Autoantibodies have been demonstrated in MS, SLE, motor neuron disease (MND), Alzheimer's disease, Downs syndrome, paraneoplastic disease and AIDS dementia. Psychosis have been described in neurolupus, MS, Alzheimer's disease, Parkinson's disease, cancer related psychosis and AIDS dementia. The relationship between Hodgkin 's lymphoma and MS, Lymphoma and MND, CNS lymphoma and HIV infection and lymphomatous transformation in SLE and rheumatoid al'thritis have been documented. Viral persistence as an etiological factor have been documented in MS, Parkinson's disease, Non Hodgkin's lymphoma and schizophrenia. Hyperinsulinemia has been documented in Alzheimer's disease and immune mediated neuropathies described in syndrome-X. This interrelationship is possibl y dependent on increased nicotine and strychnine and reduced morphine contributing to Na+-K +A TPase inhibition.

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