Effects of inhibitors on monoamine oxidase activity in mouse brain

Pharmacologica/ Research Communications, Vol. 14, No. 5, 1982

443

EFFECTS OF INHIBITORS ON MONOAMINE OXIDASE ACTIVITY IN MOUSE BRAIN T. R. F~II, H. R. Figueroa and P. B. Yurgens Department of Pure and Applied Zoology, The University, Leeds LS2 9ET, UK and Departamento de Ciencias Basicas, Universidad Catolica de Chile, Casilla 617, Talca, Chile. Received in final form 16 February 1982

SUMMARY Mouse brain monoamine oxidase (MAO) activity was measured fluorometrlcally following incubation with various inhibitors. Dose-response curves for clorgyline, deprenyl and harmaline were biphasic indicating the presence of two forms of the enzyme differing in susceptibility to inhibitors. Further investigation into the characteristics of inhibition showed that two phases could be distinguished; a reversible inhibition shown by harmaline at all concentrations and by clorgyline and deprenyl at low concentrations with short incubation times, and a presumably irreversible inhibition (inactivation) shown by the latter two drugs at higher concentrations and with longer incubation times or following preincubation of the enzyme with the inhibitor before the addition of substrate. LineweaverBurke analysis revealed that low concentrations of harmaline non-competitively, whereas pargyllne competitively, inhibited MAO activity when using kynuramine as the substrate. These results suggest that the drugs first inhibit MAO via reversible, non-covalent interactions, and inactivation of the enzyme is a relatively slow process delayed when substrate is also present. INTRODUCTION There have been numerous reports that monoamine oxidase (monoamine : 02 oxidoreductase, deaminating; ECI.~.3.4, MAO) may exist in brain and peripheral tissue in more than one form, each with its own substrate preferences and inhibitor sensitivities.

One form of MAO, type A, appears to oxidise

preferentially serotonin and noradrenaline and is inhibited by clorgyline and harmaline, whereas MAO type B is mot 9 specific in its actions towards benzylamine and 8-phenylethylamine and is i n h i b i t e d b y deprenyl.

The monoamines dopamine, tyramine

and kynuramine appear to be substrates for both forms of the

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444

enzyme (Youdlm, 197~;

Achee e~ a_!l., 1977;

Knoll et al., 1978).

It has been suggested that pargyllne is a type B MAO inhibitor (Squires, 1972) though this is apparently not the case ~

vltro

(Hall and Figueroa, 1981). Inhibition of MAC activity by harmaline appears to be reversible, whereas clorgyllne, denrenyl and pargyline are irreversible Inhlbltors (Burkard and Kettler, 1977; and Guha, 1978, 1979).

Mitra

However, preliminary results from our

laboratory (Hall and Figueroa, 1979) suggested that pargyllne at low concentrations was a comoetltlve inhibitor of MAO activity, i.e. acts as a reversible inhibitor.

Accordingly,

the follow~ng experiments were designed to examine the apparent "reversible" and "irreversible" nature of MAO inhlbitors on activity of MA0 in mouse brain homogenates° MATERIAL 8 AhD M~THOD8 Six month old male Swiss albino CF 1 mice were used in all experiment~.

They were maintained under a l~h bight-10h

darkness photoperlod (lights on at 06.0Oh) at 22 ± I'C with food and water provided ad libltum.

When used in experiments

they were killed by decapitation, whole brains were removed immediately and homogenized in ice-cold 0.2M glycine-NaOH buffer at pH 9.0 at a concentration of 20 mg tissue/ml buffer.

F~O

activity was assayed as described previously (Hall and Figueroa, 1981). Exoerlmental Drocedurgs:

Mouse brain homogenates were incubated

for 30 min at 37"C in buffer containing the substrate kynuramlne and either clorgyline, harmaline or deprenyl at concentrations ranging from Io-IOM to IO-~M.

A second incubation was

Pharmaco/ogicalResea~hCommun/cation~ VoLI< No.~ t982

445

performed, with IO-9M, 10-TM or lO-5M of harmallne, clorgyllne or deprenyl, and the reaction was stopped after times ranging from 5 min to 60 min.

In a further experiment, using the same

concentrations of the inhlbitors, the enzyme preparation was preincubated at 39"C with the inhibitors for periods of time up to 60 mln before the addition of substrate, following which 1.25 x lO-BM kynuramlne was added and the incubation carried out for a further 30 min.

In the final incubation, carried

out for 30 min, brain homogenates were incubated with lO'8M harmaline or 10"TM pargyllne and kynuramine at concentrations from 1.25 x IO-~M to 6.25 x IO-6M.

After all incubations MAO

activity, expressed as production of 4-hydroxyquinoline

(~HQ)

was determined spectrophotofluorometrically. D~__~:

Clorgyline was a gift from May and Baker, Dagenham,

Essex;

deprenyl was generously donated by Professor J. Knoll,

Semelweiss Univers4ty, Budapest;

all other drugs were purchased

from Sigma Chemical Co., St. Louis, Missouri, U.S.A. Data analysis:

All results are calculated as the mean of

triplicate determinations on samples from four separate brains. Regression analyses were performed by the least squares method. RESULTS Mouse brain homogenates were incubated with kynuramine in the presence of various concentrations of harmaline, clorgyline and deprenyl and MAO activity, expressed as % yield of 4HQ compared to control, uninhibited incubations, is shown in fig.l. It can be seen that all three drugs inhibited MAO activity in a blphaslc manner.

Each double sigmold curve displayed a

distinct plateau region.

At thisposition approximately ~0%

Phar,nacological Research Communications, VoL I4, No. 5, 1982

446

l

! 0

.! s

|

tO

FIG. I.

-log I~,~tm IM)

Effects of inhibitors on mouse brain MA0 %ctlvity. Homogenates were incubated with 1.25 x lO':'M kynuramine and various concentrations of clorgyllne (solid circles), harmaline (asterisks) or deprenyl (o~en circles) for 30 min. Production of 4HQ was determined fluorometrlcally and expressed as ~ o~ control, uninhibited incubations. All results are mean ~ standard error (vertical bars) of triplicate determinations on four brains.

of total enzyme activity was inhibited by harmaline and by clorgyline and about 60% of total activity was inhibited by deprenyl. MA0 activity in mouse brain was measured following incubation or prei~cubatlon of the ehzyme with different concentrations of harmallne.

As shown in flg.2, production

of 4HQ was linear with respect to time of incubation.

Addition

of harma!Ine to incubations produced a concentration-related inhibftlon of 4HQ production, and a consistent fraction of HAO

Pharmaco/ogical Research Communications. VoL 14, No. 5, 1982 ~&_

447

,,,

I

-

--oC

E o co

INCUBATION

FIG. 2.

TIME

min.

30

PREINCUBATION

,i TIME

60

ml..

Effects of harmalino on mouse brain MA0 activity, expressed as % yield of 4-hydroxyqulnollne compared to a control incubation. Left hand panel shows effects of~ncubatlon of brain homogenate + substrste (1.25 x lO-*M kynura~ine) + ha~mallne at a~ concentration of lO-YM(9), I0-7M(7) or I0 ~M(~). Right hand panel shows effects of prelncubation of homogenate + harmallne, before addition of substrate, when the incubation was continued for a further 30 min. Results are the mean of 3 determinations on each of 4 brains. For clarity standard errors have been omitted; they ranged from I-9~.

activity was inhibited at each tlme point by each concentration of the drug.

Preincubation of mouse brain homogenates with

harmallne did not alter the inhibitory efficacy of harmaline. All concentratlons of harmallne produced the same level of inhibition whether added concurrently with the substrate or whether prelncubated for up to 60 mln with the enzyme before addition of the substrate. By contrast, a different pattern of response was seen when clorgyllne (flg.3) or deprenyl (fig.%) was used instead of harmallne.

Both of these drugs produced a concentration-

Pharmacological Research Communications, I/o/. 14, No, 5, 1982

448

related inhibition of 4HQ production, which was not, however, directly related to the length of time of incubation.

With

increasing concentrations of drug, the rate of 4HQ production declined dramatically with increasing duration of incubation.

1^ -

~C g

i

•

,7

{i

///

60 INCUBATION

FIG. 3.

TIME

rnin.

PREINCUB.ATION

TIME

rain.

Effects of clorgyline on mouse brain MAO activity, expressed as % yield of 4-hydroxyquinoliue compared to a control incubation. Left hand panel shows effects of incubation of homogenate ~ substrat~ + clorgyline at a concentration of IO-WM(9), lO-/M(7) or IO-PM(5). Right hand panel shows effect of prelncubation of homogenate + clorgyline, before addition of substrate, when the incubation was continued for a further 30 mln. Results are the mean of 3 determinations on each of ~ brains.

The inhibitory effects of clorgyline and depreynl were both concentration- and time-dependent.

This pattern of response

was more pronounced following preincubation of enzyme and inhibitor.

With increasing prelncubatlon ~tlme for any given

concentration of inhibitor, production of 4HQ was reduced, and

Pharmacological Research Communications, I,/ol. 14, No. 5, 1982

449

.)_

rE

o 0

-7, 6(!

INCUBATION

TIME ~ n .

PREINCUBATION

TIME

mi~.

Effects of deprenyl on mouse brain MAO activity, expressed as % yield on 4-hydroxyquinoline compared to a control incubation. Left hand panel shows effects of incubation of homogenat~ + substr~te + deprenzl at a concentration of 10-WM(9), lO-ZM(7) o r P l O - M~5). Right hand panel shows effect of preincubation of homogenate ÷ deprenyl, before addition of substrate, when the incubation was continued for a further 30 mln. Results are the mean of 3 determinations on each of ~ brains.

also with increasing concentration of inhibitor for any prelncubation time, production of ~HQ was also reduced.

With

high concentrations of clorgyline and deprenyl the effects of

preincubatlon were rapid, with maximum response occurring in g-l~ mln, whereas with low concentrations of clorgzllne and deprenyl the effects noted were more slowly developing. Mouse brain homogenates were incubated wlth various

concentrations of kynuramlne either alone, with IO-7M pargyllne or with IO-8M harmallne.

A Lineweaver-Burke plot of MA0

activity is shown in fig. ~.

It is immediately apparent that

450

Pharmacological Research Communications, Vol. 14, No. 5, ; 982 1 4140 pM/9

H

3 P

5

15

/Sx 104 FIG. 5.

Kinetics of MA0 activity. Figure shows plot of HQ -L production vs kynuramine -L concentration following incubation of mouse brain homogenates in the presence of different concentrations of kynuramine. Results are the mea9 of determinations on 4 b~ains. C, control; P, lO- M pargyllne; H, lO-OM harmaline.

parg~llne acted as a competitive inhibitor, whereas harmallue was a non-competitive inhibitor of kynuramine deamlnation.

The exact nature of the interaction between the enzyme MAO and the drugs that irreversibly inhibit (inactivate) its activity is still not fully elucidated.

These results suggest

that the inhibitor first reversibly occupies the sensitive site on the enzyme molecule, which is presumablF an instantaneous reaction, and then interacts with the enzyme to produce irreversible inactivation, presumably by the formation of covalent bonds. is a relatively slow process.

This second phase of inhibition

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451

It is generally considered that MAO exists in two functionally distinct forms.

When tissues are prelncubated

with clorgyline, an MAO type A inhibitor, there is a doserelated reduction in MAO activity.

Low concentrations of

clorgyline inhibit serotonin metabolism, greater concentrations are required to inhibit benzylamine metabolism, and when tyramine is used as substrate a blphasic inhibition curve is seen (Johnston, 1968; Callingham, 1979).

Achee and Gaba7, 1977;

Parkinson and

Depren71, an MAO type B inhibitor, inhibits

benzylamlne deamination at lower concentrations than are required for inhibition of serotonin deamination (Knoll e_~tal., 1978).

Our results show a biphasic curve for inhibition of

kynuramine metabolism by mouse brain MAO with both clorgyllne and deprenyl and, in addition, with the reversible type A inhibitor (Burkard and Kettler, 1977), harmaline. It would be expected that a reversible inhibitor would show no time-dependent effects related to its inhibitory efficacy. This was demonstrated with harmaline (fig.2) where production of 4HQ, though reduced compared to the control incubation, was linear with respect to time for up to 60 min when harmallne was present in the medium.

Prelncubation of enzyme with harmaline

did not increase the amount of inhibition of MAO activity. This is in distinct contrast to the responses seen with clorgyline and deprenyl.

With these drugs inmubatlon of

enzyme, substrate and inhibitor resulted in production of 4HQ that was linear with time only with low concentrations of i n h i b l t o r a n d for short incubation times.

The rate of

production of ~HQ decreased with increasing incubation time, and this effect was more pronounced with higher concentrations of

Pharmaco~gicalResearchCommun~at~n~ ~ L I 4 , No.~ 1882

452

inhibitor.

Prelncubatlon of enzyme plus inhibitor before

substrate addition resulted in a loss of enzyme activity that was dependant upon both duration of preincubatlon and concentration of inhibitor.

Hence low inhibitor concentrations

produce responses typical of "reversible" Inhibitors, particularly in the presence of substrate,

i.e. the substrate

"protects" the enzy.ve from inactivation.

Further corroboration

of the reversible nature of the irreversible inhibitors is indicated by the fact that a low concentration of pargyline showed typical reversible kinetics, as did harmaline,

and

pargyline competitively inhibited deaminatlon of kynuramine. Since harmaline non-competitively inhibited kynuramlne metabolism this suggests that there are several sites of inhibitor attack on the MAO molecule.

Achee and Gabay (1977) also reported

that bovine brain mitochondrial MAO was inhibited by deprenyl with a "fast" and a "slow" phase that was time and concentration dependent and enhanced by prelncubatlon.

Figueroa e t a_!l.

(1981) and Hall et a~l. (1981) showed that pargyline in goldfish brain and pargyline, clorgyline and deprenyl in perch braln respectively showed reversible inhibiting characteristics

as

well as the expected inactivation of brain I~0 activity. ACKNOWLE~GEMENTS We would like to thank Dr. B. Unsworth for the mice, May and Baker Ltd. for the clorgyllne, Professor J. Knoll for the deprenyl and Dr. J. Brooks for the use of his spectrophotofluorometer.

This work was supported b y g r a n t s

from

Marquette University Committee on Research and BRSG ~ R R 0 7 1 9 6 to TRH, and by a Scholl Foundation Fellowship to HRF.

Pharmacological Research Communications, Vol. 14, No. 5, 1982

4 5 3

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