Sympathoadrenal activity and plasma glucose effects on plasma dopamine-beta-hydroxylase levels in rats

243

C‘lrnrca Chimrco Actu. 152 (1985) 243-252 Elsevier

(‘CA 03313

Sympathoadrenal activity and plasma glucose effects on plasma dopamine-beta-hydroxylase levels in rats J.A. Muiioz ” Deptirrcrmenro

‘.*, J. Garcia-Estaii h, M.G. Salom and M.T. Miras Portugal ’

de Putologiu

Bioquimicu,

M&/KU,

Fwultud

(Received

” Depurtumet~ro

de Farologiu

de Mrdrc~rnu. C~mwwidd

h, T. Quesada

Hunw~u

de Murcru,

cd

Murcirr

’

h

Depur~~mcw~o de

(Spurn)

March 7th. 1985: revision July 3rd. 19X5)

Summary

Plasma dopamine-/3-hydroxylase (DBH) activity is a controversial index of sympathoadrenal function. In our results, the half-life of bovine DBH administered by cardiac puncture to Wistar rats was dependent on plasma glucose values, being 60 min for controls, 96 min for streptozotocin (STZ)-diabetic animals ( p < 0.02) and 33 min for insulin-treated normal rats ( p < 0.01). In experimental situations with low plasma glucose levels, DBH activity was also diminished with respect to controls (glucose: 103.6 f 2.2 mg%, DBH: 9.7 + 0.5 U/ml). After fasting. glucose was 60.8 _t 1.5 mg% ( p < 0.001) and plasma DBH 6.4 + 0.3 U/ml ( p -c 0.001); fasting plus cold exposure also decreased glucose (66.2 + 1.4 mgR. p < 0.001) and plasma DBH (6.7 + 0.2 U/ml; p < 0.001). In both situations, there was an increase in exocytosis from sympathoadrenal tissues; however. no increase in plasma DBH levels was observed, because plasma glucose being diminished it was unable to compete at the catabolic receptor level. When normal plasma glucose levels take place, plasma DBH is essentially constant. poorly reflecting a moderate increase or decrease in exocytosis from tissues, as was the case in our animals with 48 h of cold exposure. When chemical sympathectomy (6-OH-dopamine) or bilateral adrenalectomy was performed there was a compensatory mechanism between them. Plasma DBH does not change significantly in these situations if plasma glucose values are normal. From these results, the most important physiological influence on plasma DBH activity is the glucose plasma levels. Plasma DBH values not being a useful index of

* Correspondence to: J. Andres Muiioz. Departamento Universidad de Murcia. 30001 Murcia. Spain.

0009-8981/X5/$03.30

‘” 1985 Elsevier Science

Publishers

de Fisiologia

Humana.

B.V. (Biomedical

Facultad

Division)

de Medicina,

244

s~mpathoadr~nal considered.

activity

if. at the same time,

the plasma

glucose

tescls

arc nctt

Introduction

Dopamine-/?-hydroxylase (DBH) is a copper-containing glycoprotein. which catalizes the formation of Norepinephrine from Dopamine [l]. DBH is localized inside storage granules together with catecholamines and other substances, in chromaffin cells and sympathetic nerve endings 121. When tissues are properly stimulated. the storage granule content is released via a process called exocvtosih, and a loss in their catech(~lamines and DBH content has been described [3--&j. As the existence of DBH in plasma has been reported, [7] the d~terminatioll of plaxma DBH activity could serve as an index of sympathoadrena~ function [Gil]. Moreover. exocytusis permits the presence of DBH in plasma which. together with ita greater half-life compared to that of catecholamines, could allow more accurate information to be obtained about sympathoadrenal activity in a variety of oxperimental situations. However, no correlation has yet been found between plasma and urine catecholamine levels and plasma DBH, which suggests the existence of other regulatory factors controlling plasma DBH levels. It is necessary to bear in mind that. in ail studied species [12-141, DBH is a glycoprotein with mannose at its non-reducing end and. according to Ashwell and Morel1 [15] and Summerfield et al [16] it must be cleared through a lectin-receptor. In previous work, we have reported that catabolic clearance of plasma DBH is inhibited by high glucose tevels. or other structural-analogs such as 2-deoxy-D-glucose or n-methyl-D-mannoside. which can compete at the lectin-receptor level [17]. in the present work. we have studied experimental situations in which a decrease in glucose values can be expected (fasting, cold and insulin-induced hypoglycemia) to see if plasma DBH levels are modified and if low glucose values affect the half-life of DBH. We have also tried to determine the influence of the sympathoadrenal system on plasma DBH levels, when a change in this activity occurs. Plasma glucose and plasma and tissues DBH levels were also studied to find out if DBH serve> w ;III index of s~~mpat~~oadreI~a1 function and if glucose is the only or main physi~~i(~~ical influence on plasma DBH values. Materials

and methods

Muterids Male Wistar rats without previous manipulation have been used in this work. At the 7th wk of life, rats weighing 150 + 15 g were randomly distributed in cages (5 animals/cage) and submitted to various procedures. Except when indicated. drug administration and surgical manipulation were performed with ethylic ether. Animul

treatment

Diabetes

mellitus

induction

was carried

out by injecting

streptozotocin

(STZ.

245

Sigma Chemical Co., St. Louis, MO, USA) (65 mg/kg body wt, dissolved in 0.5 ml buffer citrate, pH 4.5) by cardiac puncture. Another group of rats were bilaterally adrenalectomized through flank incisions. These rats had 0.9% saline as drinking water. Chemical peripheral sympathectomy was performed by injecting 6-OH-dopamine (6-OH-DA) (Sigma) by cardiac puncture, at a dose of 50 mg/kg body wt. dissolved in isotonic saline, containing 0.01 mol/l HCI and ascorbic acid, 1 mg/ml, in a total volume of 0.5 ml. An equal dose was repeated after 48 h and another double dose was injected 5 days after the first [18]. Another group of animals (300 + 15 g) were introduced in a cold room (4 k 2°C) for 48 h (1 animal/cage) (cold exposure group). Animals on a 48-h fast (300 + 15 g, initial body weight) were prepared by removing food from their cages (1 animal/cage) (fasting group). Control or sham-operated animals were used, by injecting the corresponding buffer at an equal volume by the same route; when cold exposure and fasting groups were prepared. control animals were arranged, l/cage at 22 + 2°C. throughout the 48 h. Blood sampling A basal blood sample (1 ml) was obtained by cardiac puncture in a heparinized syringe; blood was immediately transferred to chilled tubes and then centrifuged in the cold. at 10000 X g for 10 min; plasma was frozen at - 20°C until DBH assay. Glucose determination 0.1 ml of blood obtained by cardiac puncture was used, after deproteinization, plasma glucose assay by means of a glucose oxidase technique [19].

for

Adrenal und spleen DBH assay Adrenal glands and spleen were rapidly dissected, weighed and homogenized with an ice-cold 5 mmol/l Tris-HCl, pH 7.3, containing 0.2% Triton-X-100 (Merck, Darmstadt. FRG). The homogenates were centrifuged, in the cold, at 27000 x g for 20 min and the supernatants assayed immediately for DBH activity. DBH ussa, DBH activity of plasma and tissues was assayed by a sensitive procedure using tyramine as substrate [20]. Briefly. tyramine is converted into octopamine by the DBH in the sample, in the presence of copper sulphate as inhibitor of endogenous inactivators of the enzyme. After incubation, octopamine is methylated by addition of [S-‘4C]adenosyl-methionine (59 ~Ci/~mol. Amersham, UK) and phenylethanolamine-N-methyl transferase (PNMT). The PNMT necessary for the second step of the reaction was partially purified by the method of Axelrod [21], as modified by Molinoff et al [22]. Its spec act was 0.46 nmol synephrine/mg per h. DBH was purified using the concanavalin A method of Aunis et al [23]. The resulting [‘4C]octopamine is extracted and the radioactivity measured by a scintillation counter. In order to overcome the effects of endogenous inhibitors of DBH. the

‘Jh

appropriate copper was selected: 16.6 and 33.3 ~mol/tuhe honiogenutes.

respcctivelh.

and 47.6 pmol/tube

volunie was also previouslv respecti\cl\;. mmol/l.

and

Internal

tested:

5 and 10 pl

blanks were assayed in duplicate replicates (intraaasa))

was 5.65

adequate inactivation

each sample. Using

optimal

concentration

for 4 min). All

for adrenal. 4.9’;

of enz~mc inhibitor:,

sample

spleen homogenates.

and

LV;I\ 0.645

bwc used as ucll

samplc~ standard\ and

in the cold ( + 4’C‘). The variability

and

pre-determined amount of a partially

hctv,een

for spleen and 4.1 “C for pl:i\ma. L\;IS further

purified bovine adrenal

tested h\ adding ;I DBH

to :I duplicate of’

these aliquots of tissue homogenates and plasma. the rcco\ct-ie\

were al~va);s greater than 9OF : data wert:

not

corrected for twovertex. Tht: fitbt 41cp

of the reaction W;I\ run for 0.5 and 1 h for tissue second step for 1 h in all case\. One unit formation

adrenal

octopamine standards containing 0.2 nmol/tube

as blank5 (with the sample heated at 9jnC

The

for

p.1 fo; plasma. The optimal tyramine

75

for adrenal and spleen

for plasma. The

of 1 nmol

plu\ma assay. reyecti\cl?:

and

of DBH

;tcti\ith

the

LV;IS defined ;I\ the

octopatnine. mcasurcd 3x [‘J(‘]s~ncpl~rin~,~h

incuh~rtion

at

37°C. To

determine

the cnr\mc turnover.

total cnz>mc actiGtit.s ~olutw

obtained

at all rquired

was not considered

iii_jectcd in a single treatment).

acti\ it\ \IIIUC\ wet-c whtractetl

fron1

It i\ neccaar> to point out that the

hv cardiac puncture (in these experiment\ 0.2 0.3 ml for each ~tme)

implied onI\ 21weak dilution il I1 d

haul timcs.

control

in ewvme acti\.itv when \aacular ~.oIumt’ \\;I\ rc\torcd.

significant.

dost3 in ;I wliitii~ and

insulin-inducccl

Exogenous of

1 ml

hovinc into

Ii?pogl>ccniic

DBH

(725

i ,‘ml)

STZ-diahctic rat\ (6 I~ .I.

M;I\

(1 \\I\ ;iftt‘r I~t1tc‘~~‘~rnittial.

i%ovo. C‘openhagen. Denmark). For statistical Valitr~

evaluation the analysi\ of \.ariance and

art’ c\prcssed ;is the mean *

indicate

;I

lwbt-squaw

significant

Student’~

SI:M. .A 11 ~‘aluc .’

diffcrcncc. 1,inear rcgreaion

tiiethc)ds using an Olivetti-I’-6066

I

test

wt’rc

uwd.

0.05 \I;I\ cc~n~idcwtl to

qu:ttion\

\\cre calculated h\ the

calcul~ttor uith

;I linear rqt-ci\ton

progra ni. Result\

Plasma DBH

turnover

\\:I\ s1udied using purified

ew~mr

frc>m bovine adrenal

medulla. Plasma disappearance of this heterologous entyme is shop 11 in Fig. I. At the same time, plasma glucose levels were determined and onI> animals in the range were accepted: control animals close to 100 mg$. STZ-diabetic animals higher than 350 tngC;;and normal animals treated with insulin (6 U.I.) lower in these experimental than 50 mgc”r glucose. Half-life disappearance for DBH conditions was ahout 90 min for controls. 96 min for ST%-diabetic rats ( p ( 0.02)

correct

and

33

min it1 insulin-treated

rats ( p < 0.01).

C‘otwlutiotl ~~‘pl~~.rr~~u1)HI-I clc,tic>it.rwith plrr.smtr gluc~o.ce rw1~rr.s Table I compares mean plastna levels of DBH to corresponding values in various groups of rats under different

experitnental

plasma gluco~c

conditions.

Significant

247

Fig. 1. Serum disappearance

described diabetic

in ‘Material

curve of bovine DBH in W&tar rath. Serum

and Methods’:

n. number

of animals.

a. Control

DBH activity

was determined

as

(II = 6); A. insulin (n = 6); it.

(n = 6).

changes in one of these parameters usually corresponded to significant changes in the other in the same sense. in differing situations such as fasting, cold exposure plus fasting, and diabetes. In cold exposure no significant decrease in plasma glucose values were found, although plasma DBH levels were lower. This could indicate that other factors should be taken into account, therefore tissue levels of DBH have been studied. Adrenul,

spleen and plusmu

Table

TABLE Plasma

II shows plasma

DBH

actirlir?$ in sererui experimental

and tissue levels of DBH. All animals

conditions

were 3 mth old, this

I DBH activity

and glucose values under experimental

conditions

Groups

Plasma ~ DBH (U/ml)

Plasma glucose (mg %)

Control (rt = 8) Fasting (n = 8) Fasting + cold exposure Cold exposure (17 = 8) STZ(,I =X)

9.715 + 0.478 6.450+0.290 *** 6.730+0.180 *** 7.819~0.286 * 56.5 f 1.9 ***

103.67 + 2.21 60.84-t 1.55 *** 66.24k 1.44 *** 107.98 + 4.25 375.7* 19.4 ***

(n = 8)

One Unit (U) is the formation of 1 nmol of octopamine/ml per h. STZ. streptozotocin: II. number. * p i 0.05 vs. control group: ** p < 0.01 vs. control group; *** p -c 0.001 vs. control group.

age-control being necessary since tissue and plasma DBH levels arc both age-dependent [24]. These experiments were carried out in a short period of time (4X h). decroa~ea in content being deduced by increases in exocytosis. since there was no time for an adaptative process of synthesis which needs chronic stimulation [5.6.X]. Animals submitted to 48 h fasting presented a significant decrease ( p < 0.01) in their DBH adrenal and spleen content. though without an increase in plasma DBH activity which, instead. decreased ( p < 0.001) as did glycemia also ( p c 0.001. Table I). With fasting plus cold exposure. tissue levels of DBH diminished both in spleen and adrenal and. although an increase in plasma could be expected due to the increased release. plasma DBH levels. in fact. decreased ( 11< 0.001) to the S;IIIIC degree as had glucose ( p < 0.001. Table 1). In cold exposure, although no glucose changes were observed. plasma DBH Iecels diminished slightly ( p < 0.05). This decrease could be explained by the low exocytotic release from the adrenal glands. the DBH content of which increase ( p c 0.01). On the other hand. with fasting plus cold exposure a sympathoadrenal dissociation with respect to modification of tissue DBH levels can be observed. These experiments. however. do not provide information regarding the contribution 01 each system to plasma DBH levels and. in this way. experiments of adrenalectomy and chemical sympathectomy were carried out. Origin

of plusn~u DBH

These experiments were carried out with younger animals than in previous groups (Table I and II). Animals. 7 and 8 wk old. presented higher plasma and adrenal DBH. but lower values for spleen DBH than 3-mth-old rats. In order to find the contribution from sympathetically innervated tissues to plasma DBH, spleen was chosen as representative (Table III). Neither adrenalectomy nor sympathectomy changed plasma DBH levels with respect to control or its own group before treatment. neither were changes in glucose values observed in either group. However, when bilateral adrenalectomy and chemical sympathectomy were performed simultaneously. a dramatic decrease in plasma DBH could be

249

TABLE Plasma

III and tissue DBH activity

in several situations

Groups

Control ( n = 15) Adrenalectomy (n = 13) Peripheral sympathectomy (n = 12) Adrenalectomy + peripheral sympathectomy (n = 6)

n. number

of animals.

Plasma DBH

Plasma DBH

W/ml) 7th wk

(U/ml)

13.2kO.8 13.4kO.9 15.3 + 1.3 12.5kO.7

13.9 *0.x 11.2kO.6 12.3* 1.2 3.4kO.4 **

* p c 0.001 vs. control:

8th wk

Adrenal (U/pair 8th wk

DBH glands)

373.96 i_ 25.29 200.87 + 25.68 * -

Spleen DBH (U/mg tissue) 8th wk 0.230 + 0.003 0.111~0.011 * 0.026 i 0.002 * 0.006 + 0.004 *

** p < 0.001 vs. 7th basal sample

observed ( p < O.OOl), and no significant changes in glucose were found. In the same way, when this group was treated with STZ, glucose levels one week later increased four times above those basal values (97.3 + 3.9 to 390.9 + 48.2 mg%), and plasma DBH levels were five-fold higher (3.4 f 0.4 to 16.6 &-0.4 U/ml). These results help indicate that glucose is an important factor controlling plasma DBH levels. Regarding tissue DBH levels, a significant decrease in spleen DBH ( p < 0.001) could be measured one week after bilateral adrenalectomy having been carried out. When chemical sympathectomy is performed (its efectiveness being measured by the residual spleen DBH activity, about 10%). the adrenal gland significantly diminishes its enzyme activity (p < 0.001). These results indicate that both the adrenal gland and the peripheral sympathetic system contribute to plasma DBH levels without important changes when only one of them is suppressed, which suggests that between both systems a compensatory mechanism exists. Discussion The effect of high levels of plasma glucose on the half-life of DBH, together with increases of plasma DBH induced by high levels of glucose and other non metabolizable glucose analogs such as 2-deoxy-D-glucose. cY-methyl-D-mannoside and inuline, has been previously reported [17]. In this paper, it has been suggested an important rate for mannose/glucose/N-acetyl glucosamine/fructose receptor in the catabolic clearance of DBH from plasma which can explain the abnormal DBH values in diabetes mellitus. Plasma DBH activity can be increased in the presence of sugars which compete for the mannose lectin receptor with affinity for the glycidic moiety of DBH, too [15,26]. The degree of receptor blockade can determine the enzyme activity level. Nevertheless, no work has been undertaken to date, to find out the effect of low glucose levels on the half-life of DBH. Insulin-treated rats with 50% of normal glucose levels showed a significant decrease in the half-life of heterologous DBH, this result correlating well with the competition between glucose and DBH at the catabolic level. It should also be noted that a decrease in glucose coincides with a similar pattern in DBH, since the latter is easily cleared.

When

low

the higher ufhich

glucose

exocytotic

ditninishes)

homologous

animals

gland and spleen (the DBH content of into consider~rtion in order to understand

since.

are

In metabolic

b.ith

increase

or decrease

which

l’rotn

is not

adrenal Similarly,.

;tI;cj

as glucose

the

the

I>HH

and

conlent

in pl~ism~i

plasma

can

in

he mea-

DBH

are normal.

levels

in

system.

an

modifica-

7 hi>

interoting

;I verv

significant

decrca\c

the

slighily

lower

plasma J>BH.

increase

from

for

spleen.

I hi\

~~ttecholamin~~

u hen adrenalcctomy

arc normal

in which

where

explain

c&served

no significant

situatic)n

to ;I corresponding

s~mpathoadrenal

~YOCL totic

hecn

values

it is the onlv

animals

Gtuations

values

glucose be attributed

could

hv the

hns

cold

to aplain

althoug

can

gland

in non physiological out

plasma

exposure

adrenal

compensated

and

no incrcuw

parameter

of

in cold

the

values

situations.

parameter

dissociation

carried

important

in plasma DBH

can be observed

exocytotic

glucose

oii1y. glucose values diminish and catabolic the homologous DBH therefore being abacnt [ 171.

can he expected.

exocytotis

plasma

fasting

in which

DBH

fasting

Howewr.

and

the most

situations

of plasma

in the

both

experimental

changes tion

to

diminishe\.

glucose

is. therefore.

hc taken

exposed

as occurs between

the above-mentioned

aspect

both

[27],

the adrenal

in plasma.

tissues

competition <;lucose

by fasting

from

should

sympathoadrenal sured.

are ohtaincd

levels

DBH

When

values release

no significant

in

\\ mpatho-

turno\t’r

[28].

or sympathectom\

changes

in plasma

arc

DBH

tahe

ph2. In

;I normal

one system. from

the

tom\

with

situation.

although peripheral

mechanism

or

absence

functional

in plasmaU

pathectomired DBH

than of

of either DBH

activity

can

release depends

system

gluco.se

itself

occurs. on

gl~~cose

Since

peripheral

the

mo
;I useful levels.

unrlort~tken

Such

\\hcn

and

4) m-

plasma

\trengthen\

the

parantcter

to

II vcr!

tncrc;t~c’

(311. In most

its phvsiological

;I

;i dramatic

;I higher

;I result

important

high

c\plnin

c‘ast14. however.

cc)tnpctiti\e

catabolic lectin receptor [ 171. that In h~~matia. plasma DHH enz.yme is also ;I glycoprott5n conc;tn;tvalin A. although aluayh showes mannoa in ;I terminal could hi thought that the clearance rate can he modulate h\ in plasma is longer glucose’ in plasma. Moreo\ cr. its half-life DBH in rats [ 13.24.31]. ‘Thus plasma glucose wlues could

me Ievel.

allowing (he anatomical by the other [ZSl. I-bus

~tdrenulrctomi,~d

parameter

as in pheochr~~mocvtoma

dcri\ecl

sympatheccw!

to make them also diabetic IS registered.

of an?

ih mainI!

the plasma

to he present,

[.?O]. When

J-wing

plasma

contribution I)BH

at-t’ siiiiLtltan~ou~lv

STZ

i> only

[24].

to he compen~atcd

animals

lewla

plasma

change

found

he ohs&d \\ith

the cffectivc that

significantly

system

in control

the

plasma UBJ~ activit). J$tsma DBH acti\tty exocyt()tic

not

IS thrreforc.

rats are treated

acti\,ity

possibility

nervous

dws

and s~nipathrctcrtii~

adrenalectoniv

decrease

to e\aluatc

he considered

sympathetic

6-OH-dopamine

compensatory when

it is difficult

it can hardly

SLtgAt-

in this

at

the

totall? precipitate h> pwiticw [13]. and it the conccntraticw

01

that

or

hctcrolngcw~

homologous

cc>ntroversi;tl se\cr:il

results

physiological

when

the

human

or pathc~logical

haw

an important

plasma situations.

DBH

rc)le helping is t~~pl~~ecl

II) understand

:IS ;I pmmcter

111

251

Acknowledgements

The authors wish to thank Professor M. Canteras Jordana for his help in the statistical analysis. Ma. Jose Salazar who typed the manuscript and M. Harwood for his translation. This work was made possible by the grant no. 83/0905 from the Fondo de Investigaciones Sanitarias of the Spanish Ministry of Health. References 1 Frtedman

S. Kaufman

S. 3, 4-Dihydroxyphenylethylamine-fl-hydroxylase.

Physical properties,

copper

content, and role of copper in the catalytic activity. J Biol Chem 1965: 240: 4763-4773. 2 Potter LT. Axelrod

J. Properties of norepinephrine

storage particles of the ratheart.

J Pharmacol

Exp

Ther 1963; 142: 291-29X. 3 Phillips JH.

Apps

DK.

Storage

Biochem Phyaiol Pharmacol 4 Viveros OH,

Argueros

L. Kirshner

the adrenal medulla. Life Sci Part 5 De Potter WP. De Schaepdryver proteins

together

and secretion

of catecholamines:

the adrenal

medulla.

Int

Rev

Biochem 1979: 26: 121-17X. N. Release of catecholamines

I

Physiol Pharmacol

AF.

Moerman

with noradrenaline

and dopamine-P-hydroxylase

from

196X: 7: 6099618.

EJ. Snnth AD.

upon stimulation

Evidence for the release of vesicles

of the splenic nerve. J Physiol

1969:

204:

102 -104. 6 Kopin IJ. Kaufman

S. Viveros H. et al. Dopamine-b-hydroxylase.

Basic and clinical studtes. Ann lnt

Med 1976: 35: 21 l-228. 7 Weinshtlhoum

R. Axelrod

X ,Arnair JM. Garcia AC.

J. Serum dopamine-P-hydroxylase.

Horga FJ. Kirpekar

of various animals species after neurogenic sympathetic Y Cuheddu

LX.

Barhella

content of DBH

YR.

Marrero

A. Trifaro

4763

stimulation.

L. Serum dopamine-P-hydroxylaae

and DBH

activity

J Physiol 197X; 2X5: 515-529.

J, Israel AS. Circulating

pool and adrenal

in rats. guinea-pigs. dogs and humans: their role in determining

changes in plasma enryme levels. J Pharmacol 10 Geffen

Circ Res 1971: 2X: 3077315.

SM. Tissue and plasma catecholamines

soluble

acute stress-induced

Exp Ther 1979; 211: 271-279.

as an index of sympathetic

function.

Life Sci 1974: 240:

4773.

11 Roffman

M. Freedman

P-hydroxylase 12 Grranna

activity.

LS. Goldstein

M. The effects of acute and chronic swim stress on dopamtne-

Life Sci 1973: 12: 3699376.

R. Coyle JT. Rat adrenal dopamine-P-hydroxylase:

istics. J Neurochem

13 Mtras Portugal MT. Aunis D. Mandel

P. Studies on the interaction

various sources with phytohaemagglutinins. 14 Miras Portugal MT.

Mandel

dopamine-/3-hydroxylase.

17 Muiitw

compositions

of human serum

in the hepatic recognition

and transport of

1974: 41: 99-12X.

J. Joenes EA.

Modulation

of a glycoprotein

A. Serrano

C. Garcia-Estan

BA. Spector S. Tarver

h-hydroxydopamine 19 Werner

W.

Rey

J. Quesada

JH.

T. Miras

Portugal

HG.

M, Freedman

serum. Experientia

system on rat

Wielinger

H.

Propietes

Br J Pharmacol d’un

nouveau

MT.

Effect

hvper112~~1132. depletion hv

of diabetic

activity. Diahetes 19X4: 33:

Resistance of noradrenaline

or immunosympathectomy.

glucemia par la metode a la GOD-POD 20 Goldstein

recognition

cells hy glucose and diabetes mellitus. J. Clin Invest 19X2; 69: 1337-- 1347.

glycemia and other sugars on plasma dopamine-/&hydroxylase

1X Berkowitz

from

Res 1976: 1: 403 -40X.

Adv Enzymol

JA. Vergalla

hepatic endothelial

character-

of dopamine-a-hy~iroxylase

P, Aunis D. Amino acid and carbohydrate

Neurochem

glycoproteins.

16 Summerfield

and immunologic

Clin Chin1 Acta 1975: 64: 293-302.

15 Ashwell G. Morel1 AG. The role of surface carbohydrates circulating

purification

1976; 27: 1091~1096.

in blood vessels to

1972; 44: 10-16. crom@ne

Z Analvt Chem 1970: 252: 224

dcrtin$

;LU dosage &

I:,

230.

LS, Bonnay M. An assay for dopamine-P-hydroxylase

activity in tiscue

and

(Basel) 1971; 27: 632- 633.

21 Axelrod J. Purification 1962: 237: 1657-1660.

and properties

of phenylethanol-amine-N-methyl

transferase.

J Bml (‘hem