Short circuit theory on the onset of mental illness

Short circuit theory on the onset of mental illness

CLINICA CHIMICA ACTA SHORT LAURO CIRCUIT GALZIGNA 5 THEORY AND ON THE ANTONIO ONSET OF MENTAL A. RIZZOLI* de Recherches U-67 (INSERM), B...

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CLINICA CHIMICA ACTA

SHORT

LAURO

CIRCUIT

GALZIGNA

5

THEORY

AND

ON THE

ANTONIO

ONSET

OF MENTAL

A. RIZZOLI*

de Recherches U-67 (INSERM), B.P. 1018, 34-Montpellier (France) Institute of Biological Chemistry, University of Padova, 35100 Padova (Italy)

Grou@

(Received

ILLNESS

and

March 31, 1970)

SUMMARY

Acetylcholine is able to stabilize the autoxidation products of catecholamines by forming molecular complexes which are shown to be resistant toward further transformation to dihydroxyindoles induced by ascorbic acid in vitro. Such complexes are possibly formed in vivo after a leak which might cause a short circuit between adrenergic and cholinergic systems. The interaction documented in vitro has been observed also in vivo with experiments of pharmacological and behavioural type.

The dependance of the functioning of the mind on its molecular environment, both artificial and natural, has been stressed by Paulingl through the hypothesis that localized cerebral avitaminosis or other chemical deficiences can be the remote cause of insanity. The hypothesis explains the genesis of mental illness but does not account for the way in which the illness bursts more or less abruptly from an apparently normal situation. Recently a new chemical theory2 has been developed in an attempt at explaining the onset of mental illness. This theory follows the strategy of a purely chemical approach to the problem3 in spite of the fact that its difficulty* and the non-uniqueness of what is called mental illness6 have been underlined recently. The possibility of such a new theory, which can be referred to as the “short circuit” theory, has been foreseen after observations concerning the chemical interaction between neurochemical transmitters and neurotropics drugs?. In fact a chemical interaction between acetylcholine and norepinephrine giving rise to a fairly stable complex was found together with the chemical rearrangement of norepinephrine induced by acetylcholine in an hydrophobic medium’ with physical characteristics resembling the ones of brain homogenates. In this paper further evidences are presented for the stimulation of the catecholamine -+ noradrenochrome transformation in the presence of acetylcholine and nicotinamide in vitro, together with the proof for a direct interaction between acetylcholine and pure adrenochrome both in vitro and in vivo. * Request

for reprints to Dr. Rizzoli,

Padova. Clin. Chim. Acta, 30 (1970) 5-11

6

GALZIGNA,

MATERIALS

AND

RIZZOLI

METHODS

Acetylcholine and nicotinamide obtained from Fluka, Buchs (Switzerland), adrenochrome and norepinephrine from Calbiochem, Los Angeles (U.S.A.) and ascorbic acid from Merck, Darmstadt (Germany) were used without further purification. The solvents employed were redistilled before use. The interaction ilz vitro was monitored

spectroscopically

with a Cary 15 re-

cording spectrophotometer and a Beckman Infra-red spectrophotometer, chromatographically on silica gel plates with water-acetic acid 2% as solvent and by conductimetry with a Radiometer CDM 2 conductivity meter. The fluorescence was analysed by means of an Eppendorf recording spectrophotofluorimeter equipped with 400 nm exitation and 500 nm emission filters. The behavioural experiments were performed with a group of 60 male mice of the same strain reared in standard conditions. Their exploratory activity was tested with a board (90 x 90 cm) divided in 9 sectors containing different objects. The animals were put on the unfamiliar territory of the board for 5-min periods. The exploratory activity was scored always at the same period of the day (from 2 to 5 p.m.) in a thermostated room on the basis of the number of entries in different sectors. Adrenochrome (25 mg/kg) and acetylcholine (75 mg/kg) were injected intraperitoneally 30-60 min before the test. The compounds were dissolved in saline and only freshly prepared solutions were administered. The pharmacological experiments were carried out by means of the sucrose-gap technique according to the method described by Axelssons which gives both the spontaneous electrical and mechanical activity of guinea-pig taenia coli. The composition of the bathing solution was the same as described by Axelsson8. RESULTS

The kinetic

of noradrenochrome

formation

in a Tris-dioxane

medium

sented in Fig. I. Both acetylcholine and nicotinamide exhibit a clear catalytic on the autoxidation of norepinephrine and the initial velocity of autoxidation

is preeffect in the

presence of nicotinamide is higher than in the presence of acetylcholine. Aminochrome formation is followed by the increase of absorbancy at 497 nm. The kinetic of dihydroxyindole formation after the addition of ascorbic acid to noradrenochrome formed as in Fig. I in the presence of acetylcholine or nicotinamide is followed by means of the fluorescence emission as shown in Fig. 2. The effect of ascorbic acid was checked also on adrenochrome by using the same conditions described by Rostona. Fig. 3 shows that the presence of acetylcholine inhibits the adrenochrome reduction at an extent which is similar to the one observed already for noradrenochrome7 whereas nicotinamide is uneffective on this process. The direct interaction between acetylcholine and adrenochrome at the excited state is shown in Fig. 4 which documents the spectral changes induced on the visible and ultra violet absorption of adrenochrome in chloroform. With such solvent the interaction is as evident as in the Tris-dioxane whereas in water or other strongly ionizing solvents no interaction occurs. At I : IO molar ratio between adrenochrome and acetylcholine a hyperchromic effect on the 497 nm band of adrenochrome is evident; it becomes hypochromic as the molar ratio approaches I : I. Clin.Chim. Acta, 30 (1970) 5-11

THEORY ON ONSET OF MENTAL ILLNESS

Fig. I. Autoxidation B, Norepinephrine+

7

of I x 10-3 M norepinephrine in Tris-dioxane (I : IO). A, Norepinephrine alone; I x IO-~ M acetylcholine; C, Norepinephrine+ I x 10-l M nicotinamide.

Fig. 2. Dihydroxyindole formation in the presence of ascorbic acid. A, I x ro+ M Norepinephrine. z ml, I x IO-~ M acetylcholine, 0.6 ml, 1% ascorbic acid, 0.02 ml; B, I x IO-~ M Norepinephrine, z ml, Tris-dioxane. 0.6 ml, I o/0 ascorbic acid, 0.02 ml; C, I x 10-s M Norepinephrine, 2 ml, I x IO-~M nicotinamide, 0.6 ml, I o/0 ascorbic acid, 0.02 ml. All solutions were prepared by using Tris-dioxane as a solvent.

j;.ll;-;,:;_, t 1.5

I

0.5

I ,

I

1.5

Time (mini

I

2

300

350

‘.._._._.___.-_._

300

._._._

350

-

._._.

_.-._.

400 450 500 wavelengtn(nm)

550

600

Fig. 3. Reduction of adrenochrome by 2.4 x IO-~M ascorbic acid according to RostorP. A, 0.5 x IO-~ M Adrenochrome-tj x IO-~ M acetylcholine; B, 0.5 x IO-~ M Adrenochrome; C, 0.5 x 10-3 M Adrenochrome + 5 x IO-~ M nicotinamide. Fig. 4. Adrenochrome-acetylcholine interaction in chloroform. Lower part: A, 2.5 x IO-* M Adrenochrome; B, 2.5 x IO--~ M Adrenochrome+ I x IO-* M acetylcholine (Cl-) ; C, I x IO-* M AcetylB z ----; C = ._.-, choline. A = --; Upper part: Difference spectrum with double compartment cells containing mixed adrenochrome and acetylcholine above and separated adrenochrome and acetylcholine below. The solutions A, B, and C were diluted I : IO with chloroform. All measurements were made I h after preparing the mixtures.

The effect of different concentrations of acetylcholine on the 497 nm absorption of adrenochrome yielded linear log-log plot being a proof for the complex formation according to the method described by Brealey and KashalO. From this plot an apC&z. Clim. Ada, 30 (1970) 5-11

8

GALZIGNA. RIZZOLI

parent association constant of about I x 10-s M was calculated which differs from the acetylcholine-noradrenochrome interaction in Tris-dioxane2 by a factor of IO. This constant calculated for the can be due to the competitive effect of the Tris-dioxane solvent and indicates the possibility that electrostatic-type forces play an important role in the acetylcholine-aminochrome interaction. Fig. 5 shows the I.R. spectra documenting a general decrease of band transwove r!“rnbW 3000

2500

2000

cm-’ 1500

1000

100

Fig. 5. Infra red spectra in chloroform. 0.1 M Acetylcholine versus chloroform (----) and acetylcholinei-0.025 M adrenochrome versus adrenochrome (---). The spectra were taken I h after the preparation of solutions.

mittances of acetylcholine induced by the presence of adrenochrome. This can be interpreted as due to mutual polarization of the adrenochrome and acetylcholine molecules and cannot be an atypical effect since the relative ratios among the different bands are also changed. Table I summarizes the chromatographic and conductometric data showing an interaction at the ground state of adrenochrome with both acetylcholine and nicotinamide. A decreased chromatographic mobility and a non-additive conductivity both point to an electrostatic type interaction between acetylcholine and its partners in the low conductivity medium used for the assays. TABLE

I

ADRENOCHROME-ACETYLCHOLINE

INTERACTION

AT

THE

GROUND

STATE

Conductivity measurements were made at 20’ with the CDC I 14 cell of a conductivity meter type Radiometer CDM2. Chromatography was carried out on Eastman sheets 20 x 20 type K 301 Rz. 20 ~1 of each solution were used and the spots were detected by fluorescence with 3600 A U.V. lamp after spraying the plates with 10% methanolic KOH. Comfiounds

Tris-dioxane

in the solvent

Rf (water-acetic

acid

Acetylcholine I x 10-s M Adrenochrome I x IO-* M Nicotinamide r x IO-~ M

0.86 -

Adrenochrome-acetylcholine Adrenochrome-nicotinamide

o.73*

0.71

2%)

Conductivity (micro-Siemens)

2.9 2.4 3 2.5 2.6

* A second spot with Rf 0.58 was observed which can be identified as adrenolutin. Clin. Chivn. Acta, 30 (1970) 5-11

THEORY ON ONSET OF MENTAL ILLNESS

9

The experiments have been repeated by using choline instead of acetylcholine and dopachrome instead of adrenochrome and the same types of interaction have been obtained. The behavioural results, summarized in Table II, show that adrenochrome alone causes only a relative change in behaviour statistically non significant, while the contemporary administration of acetylcholine and adrenochrome induces a statisTABLE

II

BEHAVIOURAL

EXPERIMENTS

The exploratory

activity

Treatment Saline Adrenochrome Adrenochrome-acetylcholine Acetylcholine

ON MICE

is measured as given in the text. Number of animals

Mean of entrances

Signi$cance of difleerences with control (t test)

18

13&z *o f 3 7fZ I5 + 4

n.s. P = 0.05 n.s.

I4 18 IO

tically significant decrease of higher nervous activity. In other words a true psychotomimetic activity is found11 only with acetylcholine and adrenochrome and this can be related to the observation that adrenochrome introduced intraventricularly13 induces a decrease of motor activity. Acetylcholine may act either by increasing adrenochrome penetration or by a true synergic effect. Fig. 6 shows the contractile effect of I x IO-~ M acetylcholine associated with

Fig. 6. Guinea-pig taenia coli. Upper record: mechanical activity. AC = I x IO-’ M acetylcholine (Fluka, Buchs) and AD I x IO-~ M adrenochrome (Sigma, St. Louis) + I x IO-’ M acetylcholine. Lower record : electrical spontaneous activity.

an irregular membrane potential change. If I x IO-~ M adrenochrome is given together with acetylcholine the mechanical activity is absent, the muscle relaxes and the electrical activity appears as hyperpolarization. The interaction between acetylcholine and adrenochrome results in a dissociation between electrical and mechanical activity. Cl&. Chim. Acta, 30 (1970)

5-11

GALZIGNA,

IO

RIZZOLI

CONCLUSION

The present data document the influence of acetylcholine and nicotinamide on aminochrome formation, the inhibitory effect of acetylcholine on the ascorbic aciddependent adrenochrome reduction and the direct interaction between acetylcholine and adrenochrome both in vitro and in vivo. The spectral data suggest the possibility

of an interaction

of the O-C-C-N

systems of acetylcholine and adrenochrome molecule in a complex held mainly by electrostatic forces and in general all the observed effects are interpretable in term; of association eff ects13. A tentative formulation

of the type of interaction must consider the conformational adaptivity of acetylcholine14 which should be bound by its muscarinic side in a hypothetic I:I complex with adrenochrome. This is in agreement with the pharmacological studies documenting a block of muscarinic effects of acetylcholine by adrenochrome and with the data obtained in vitro on the reduction by ascorbic acid. In fact an electrostatic interaction with acetylcholine can prevent the proton removal and the series of electromeric shifts at the C, and C, atoms of adrenochrome which bring about the transformation to dihydroxyindole by effect of ascorbic acid through polarization of the C, and C, groups15. The demonstrated ability of acetylcholine

and nicotinamide

in forming mole-

cular complexes with adrenochrome, which differ in their behaviour toward ascorbic acid reduction, can be related with the fact that different groups of mental patients are able to eliminate a lower amount of ascorbic acid than normals when submitted to a loading test (L. Pauling, personal communication). This might reflect a suboptimal functioning of ascorbing acid-dependent enzymatic processes as postulated by Paulingl, but can also indicate a greater need for ascorbic acid due to the probable existence in patients of stabilized psychotogen aminochromes which need to be eliminated. The results reported here allow to explain the observed inhibition of acetylcholinesterase by adrenochrome16 and relate it to a blocking of the substrate rather than to a direct inhibition of the enzyme. In fact adrenochrome might block the site of acetylcholine molecule which should be free for the nucleophilic attack of water that causes the ester to be hydrolysed. Further work is now in progress to ascertain the type of interaction of adrenochrome with nicotinamide and to solve the chemical problems arising from the study of the in vitro systems. ACKNOWLEDGEMENT

The financial support of Professor Linus Pauling of Mr. R. De Pirro are gratefully acknowledged.

and the technical

assistance

REFERENCES I z 3 4

L. L. A. A.

PAULING, Science, 160 (1968) 265. GALZIGNA, Nature, 225 (1970) 1058. HOFFER AND H. OSMOND, The Hallucinogens, Academic Press, New York, J. MANDELL AND C. E. SPOONER, Science, 162 (1969) 1442. 5 C. P. ROSENBAUM, J. Nervous Mental Disease, 146 (1968) 103.

cht.

Chit?‘&ACta, 30 (1970) 5-11

1967.

THEORY

II

ON ONSET OF MENTAL ILLNESS

6 L. GALZIGNA, Biochem. Pharmacol., 18 (1969) 2485. 7 L. GALZIGNA, Compt. Rend., 268 (1969) 2498. 8 J. AXELSSON, J. Physiol. (London), 158 (1961) 381.

g IO II 12

S. ROSTON, Nature, 194 (1962) 1079. G. J. BREALEY AND M. KASHA, J. Amer. Chem. Sot., 77 (1955) 4462. S. GROF, M. VOJTECHIOVSKY, V. VITEK AND S. PRANKOVA, J. Neuropsychol., 5 (1963) 33. W. S. MCCUL~OCH, J. Nervous Mental Disease, 119 (1949) 271. r3 J. R. DYER, Applications of Absorption Spectroscopy of Organic Compounds, Found, Modern

Organic Chemistry Series, Prentice-Hall, Inc., Englewood 14 C. CHOTHIA, Nature, 225 (1970) 36. 15 R. A. HEACOCK, Aduan. Heterocyclic Chem., 5 (1965) 205. 16 H. WAELSCH AND H. RACHOW, Science, g6 (1942) 386.

Cliffs, N.Y.,

1965.

Clin. Chim. Acta, 30 (1970) 5-11