Catecholamines: Strain differences in biosynthetic enzyme activity in mice

Life 3ciencee Vol. il, Part I, pp. 585-572, 1972 . Printed in Great Britain

CATECHOLAMINES :

Pergamon Press

STRAIN DIFFERENCES IN BIOSYNTHETIC ENZYME ACTIVITY IN MICE

Roland D . Ciaranello, Rebecca Barchas, Seymour Kessler and Jack D. Barchas Department of Peyahiatry, St~foztil University SohooZ of Medicine Stanfor<3, California 94305

(Received 10 December 1971 ; in final form 20 Aprll 1972)

In five strains of inbred mice, activities of phenylethanolamine N-methyl transferase and tyrosine hydroxylase and amounts of norepinephrine and epinephrine in the adrenal gland vary markedly . Tyrosine hydroxylase activity in the brain also varies widely . The fact that these measures vary independently of each other suggests that several genetic factors are involved . The finding of genetic differences affecting cetecholamine synthesis has implications for the study of catecholamine regulatory mechanisms and the relationship between catecholamines and behavior . The catecholamines epinephrine and norepinephrine are compounds with important biological functions .

Epinephrine, the principal hormone secreted

by the adrenal medulla, is formed from norepinephrine by the enzyme phenyleth anolamine N-methyl transferase [PNMT](1) .

Norepinephrine functions as the

neurotransmitter in the adrenergic nervous system and as a neuroregulatory agent in the brain (2,3) .

The catecholamines have received widespread atten-

tion because of their possible association with mental disorders (4,5,6) and with diseases of the cardiovascular and other systems (7) . Strain and subline differences have been reported in the amounts of biogenic amines in brain regions of mice (8,9,10,11,12) and rats~~13,14) and in the utilization and uptake of cardiac norepinephrine in mice (15) .

To

gether with the known genetic variation in adrenocortical function (16,17, 18) and in adrenocortical and adrenamedullary structure (19,20), these findings suggest that the search for genetic variation in the enzymes involved in cntecholamine biosynthesis would be fruitful .

lie report here strain differences

in the activities of PNMT and tyrosine hydroxylase (TH), two enzymes involved in catecholamine synthesis.

TH is involved in the first and rate-limiting

step in catecholamine biosynthesis, converting tyrosine to dihydroXyphenylal-

585

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Düierences in Catecholamines Synthesis

anine .

Vol . il, No. 12

A preliminary report of genetic variation in adrenal PNMT and TH and

brain TH in mice has been published elsewhere (21) . Materials and Methods We studied males of five inbred mouse strains, BALB/cJ, C57BL/Ka, C57BL/IOJ, CBA/J and DBA/2J, all purchased from the Jackson Laboratory, Bar Harbor, Maine, except C57BL/Ka which was obtained from the Department of Radiobiology, Stanford Medical Center .

All animals were 6 to 8 weeks old at the time of the

study, with a mean weight of 22 .7 t 0 .4 g .

Animals, housed six to a cage with

free access to Purina Chow and water, were maintained on a cycle of 13 hours of light and 11 hours of dark for at least 4 to 5 days before being killed by decapitation . ice .

Adrenals and brains were removed and frozen immediately on dry

The adrenals of six animals in each strain were assayed for PNMT or TH

activities ; individual adrenals were randomly assigned to one assay or the other .

Each assay was done on the adrenals of the five strains of mice on the

same day .

Adrenals were homogenized in 0 .3 ml of ice-cold 0 .32 M sucrose .

Homogenates for PNMT determinations were centrifuged at 30,000 g for 30 minutes, The PNMT activity was measured by

and 100-u1 portions were taken for assay .

the method of Axelrod (1), using phenylethanolamine as substrate and S-[methyl14 C]adenosylmethionine as methyl donor .

The small amount of radioactive metha-

nol formed enzymatically as an interfering product was removed by the procedure of Deguchi and Barchas (22) .

After extraction of the N-[methyl- 14 C]phenyl-

ethanolamine into toluene isoamyl

alcohol

(97 :3), 4 ml of the organic phase

was extracted into 1 ml of 0 .1 N HC1 and 0 .5 ml portions of the acid-phase were taken for scintillation counting . were used as blanks .

Samples from which substrate was omitted

The TH activity was measured by the method described by

Dr . M . Levitt who was kind enough to send us a preliminary draft of his manuscript, now in preparation .

The sucrose homogenate was centrifuged at 10,000 g

for 20 minutes and a 50 ul portion of the supernatant was assayed with l 4 Ctyrosine (Miersham-Searle, 507 mCi/mmole, uniformly labeled) as the substrate .

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Ditferences in Catecholamines 3y~hesis

587

Boiled enzyme was used in the blanks ; the radioactivity in these blanks was slightly less than in blanks from which enzyme was omitted .

For determination

of TH activity, Incubation with the following reagents was carried out (all concentrations being in micromoles/reaction tube, final reaction volume was 500 microlites) : potassium phosphate buffer (pH 6), 100 ; mercaptoethanol, 100 ; NSD-1055, 0 .1 ; 2-amino-4-hydroxy-6, 7-dimethyltetrahydropteridine, 1 .0 ; L-[ 14 C]tyrosine, 0 .3 uCi .

Samples were incubated at 37°C for 15 minutes, after which

0 .1 ml of 25 percent trichloroacetic acid was added .

Samples were transferred

to tubes containing 50 ug of L-DOPA and centrifuged at 17,000 g for 10 minutes . To the resulting supernatant 5 ml of 0.2 M sodium acetate, 0 .2 ml of 0 .2 M EDTA, and 0 .3 g of alumina was added .

The sample was titrated to pH 8 .6 with

1 N NHyOH, stirred for 3 minutes and poured as a slurry over a column packed with glass wool .

The columm was washed with 30 ml of water, and the L-[ 14 C]-

dihydroxyphenylalanine was eluted with 6 ml of 0 .3 M acetic acid . Results and Discussion Fig . 1 shows the mean (t S .E .) adrenal PNMT and TH activities for six animals in each strain .

Significant differences (p < .O1) between the strains

were shown by analysis of .variance .

For each enzyme, the BALB/cJ strain had

high whereas the C57BL/Ka strain had consistently low activities . Six additional animals of each strain were killed to determine adrenal catecholamine content by the method of Barchas et al . (23) using a Bio-Rex 70 resin for extraction of the catecholamines . Table 1 .

The results are shown in

The differences between strains, assessed by analysis of variance,

were significant (p < .O1) for both catecholamines .

The difference in epineph-

rine content between the highest and lowest strains was two-fold ; that for norepinephrine was somewhat less .

588

DiSerences in Catecholamines Synthesis

PNMT

Vol. 11, No . 12

Lso L60

L

.l4

1.~0

.12

1 .20

.lo

L00

â w L

w 0

.60 A4 .02 Ao

u

u

â

FIGURE 1 . PNMT activity is expressed as the nuaber of millimlcrovloles N-[methyl- 1 "C]-phegylethanolamine formed per hour per adrenal . TH activity is expressed as the amount of DOPA- 1 `C fornled per hour per adrenal . Mean (t S .E .) adrenal PNMT and TH activities in inbred arouse strains (6 animals per strain) .

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TABLE 1 Adrenal Weights and Adrenal Catecholamine Levels in Inbred Mouse Strains (means

Strain

t

S.E . of 6 animals)

Adrenal Weight (ma)

Concentration (u9/mg of adrenal)

Left

Norepinephrine

Epinephrine

Righ t

BALB/W

1 .84 t 0.08 1 .25 t 0.07

1 .18 t 0.11

1 .55 t 0.15

C57B1/Ka

1 .57 ± 0.07

1 .09 t 0 .05

0 .94

0.10

1 .17 t 0 .07

C57B1/10

1 .78 t 0.10

1 .31 ± 0.10

0.99 t 0.04

1 .19 t 0.06

CBA

1 .57 ± 0.07

1 .52 t 0.05

1 .40 ± 0.07

2 .02 t 0.13

DBA/2J

1 .43 t 0.10

1 .03 t 0.11

1 .41 t 0.17

2.43 t 0.16

f

Although the correlation between the amounts of epinephrine and norepinephrine was significant (p < .05), when all strains are considered, no obvious relationship between enzyme activities and catecholamine stores was apparent . This is not surprising since other factors besides catecholamine synthesizing enzyme activity (e .g ., rate of release from the medulla, endogenous turnover, etc.) affect adrenal catecholamine storage pools. Although, as expected (2), there were significant (p < .O1) strain differences in adrenal weight (Table 1), it is doubtful whether this factor, itself , could explain the variation in adrenal catecholamine content and zyme activities reported here .

en

The statistical significance of the interstrain

differences in catecholamine content and their relative rank order are not influenced by the expression of the data as total micrograms or as micrograms per milligram of adrenal tissue .

Also, no association between adrenal weight

and relative enzyme activity was apparent . mean

In the BALB and CBA strains, the

adrenal weight did not differ ; the fornier showed high PN+IT and TH activi-

ties whereas the latter showed relatively low values of both adrenal enzymes. The possibility remains that differences in adrenomedullary volume (2) might explain same of the lnterstrain differences in enzyme activities .

However,

in the C57BL/Ka and DBA strains, both with relatively small adrenals and

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relatively low TH activities on a per adrenal basis, the latter strain showed more than twice as much PNMT activity as the former .

These findings strongly

suggest that among the interstrain enzyme differences reported here, some or all exist independent of any morphological differences between the adrenal glands of the various strains .

We believe that the variations reported here

are due either to differences in the concentration and/or molecular structure of the pertinent enzymes . Interest in catecholamines as neuroregulators or neurotransmitters in the brain led us to determine the activity of TH in the brains of the different strains .

Tissues were homogenized in four volumes of ice-cold sucrose in a

Polytron (Brinkman Instrument Co .) generator, a unit which also acts as a low energy sonifier .

Of the measurable TH in brain, 96 percent was liberated into

the supernatant .

Centrifugation was carried out as described above and 0 .5 ml

portions of the supernatant were assayed .

The results are shown in Table 2 .

TABLE 2 Brain Weights and Brain TH Activities in Inbred Mouse Strains (means t S .E . of 6 animals)

Strain

Brain Weight

Brain TH

(g)

~molesjhrJ~

BALB/cJ

0 .4456 t 0 .0128

20 .09 t 2 .79

C57B1/Ka

0 .3439 t 0 .0116

15 .66 t 2 .00

C57B1/10

1 .4152 t 0 .0090

10 .76 ± 0 .54

CBA

0 .4166 t 0 .0251

10 .03 t 0 .53

DBA/2J

0 .3532 t 0 .0092

10 .98 t 0 .67

Malysis of variance showed the presence of significant (p < .O1) strain differences .

The relative rank order of strains was identical for both adrenal

and brain TH with the notable exception of C57BL/Ka, which showed the lowest adrenal TH in combination with a relatively high activity of brain TH .

This

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Differences in Catecholaminea S~ntheaie

571

finding raises the possibility that structurally different forms of this enzyrt~e and/or differences in the rates of turnover exist in the two tissues . Summary We have investigated the activity of catecholamine synthetic enzymes in vitro and, for the first time, have presented evidence suggesting that genetic variation affecting the activities of these enzymes is present in adult mice . Such variation could be exploited in the study of the induction and regulation of these enzymes, in the search for different enzyme forms, and in the exploration of the release, uptake, utilization, metabolic pathways, receptor effects and turnover of the catecholamines in their target tissues in vivo .

Genetic

variation in any of the preceding aspects involving catecholaminés may have a direct bearing on the relationship between catecholamines and psychopathological states . References 1.

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2.

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W .E . Bunney, Jr ., H .K .H . Brodie, D . L . Murphy and F . K . Goodwin, Amer . J . Psychiat . , 127, 7 (1971) .

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Vol. il, No . 12

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23 .

J . Barchas, E . Erdelyi and P . Mgwin, Mal . Biochem . , in press . This work was supported in part by PHS Grant MH 16,632 . recipient of NIMH Bio-Science Grant MH 8304 . Scientist Development Award MH 24,161 .

RDC was a

JDB holds NIMH Research