Mechanisms of catecholamine accumulation in adrenal chromaffin granules

Brain Research, 62 (1973) 317-326

317

© Elsevier ScientificPublishing Company, Amsterdam- Printed in The Netherlands

MECHANISMS OF CATECHOLAMINE ACCUMULATION IN ADRENAL CHROMAFFIN GRANULES

A. PLETSCHER, M. DA PRADA, H. STEFFEN, B. LI~TOLDANDK. H. BERNEIS Research Department, F. Hoffmann-La Roche and Co. Ltd., Basel (Switzerland)

The catecholamines (CA) released from catecholaminergic nerve terminals into the synaptic cleft by nerve stimulation are thought to be inactivated mainly by re-uptake into the terminals. During this re-uptake process, the amines have to pass the synaptic membrane of the nerve terminal and to enter the presynaptic organelles storing the amines. The transport of the amines across the synaptic membrane probably occurs by an active, carrier-mediated mechanism which is blocked by drugs like ouabain and imipramine. The storage process in the presynaptic organeUes involves transport through their membrane and probably also a second mechanism by which the amines are retained within the organelles. This presentation deals with the storage process of CA in chromaffin granules of bovine adrenal medulla. These organelles have been chosen since they can be isolated in virtually pure form and might serve as models for other amine-storing organelles, e.g. of catecholaminergic nerve terminals. Two main questions were studied, i.e. the uptake of the amines at the level of the membrane of the storage organelles and the physicochemical form in which the amines are present within the organelles. Uptake by granular membranes

The uptake mechanism of CA into storage organelles has been studied by using suspensions of isolated storage organelles. However, this technique does not easily differentiate between the transport at the level of the granular membrane and the intragranular storage mechanism. Therefore, in order to elucidate the role of the granular membrane in the storage process, it is preferable to use preparations of isolated granular membranes. Evidence has previously been presented that membrane preparations (obtained by osmotic shock of isolated granules) consist chiefly of membrane-enclosed empty vesicles which are probably newly tbrmed from membrane fragments. These membranes, which contain a Mg2+-dependent adenosine-5'-triphosphatase (ATPase) 20, take up monoamines, whereby noradrenaline has been shown to be transported from the incubation medium into the vesicles1,2t. The amine uptake exhibits several characteristics, some of which can be summarized as follows.

318

A. PLETSCHER et al.

TABLE 1 UPTAKE OF VARIOUS RADIOACTIVE AMINES BY ISOLATED MEMBRANES OF BOVINE ADRENAL CHROMAFFIN GRANULES INCUBATED IN AN ARTIFICIAL MEDIUM 21 AT

37 °C

FOR 15

rain

Initial concentration: amines 45/~M, ATP 5 raM. The figures show averages with S.E. of 3-7 experiments and are indicated in nmole~/mgprotein 14. 5-HT = 5-hydroxytryptamine. Amine

Without ATP

With ATP

5-HT Dopamine Adrenaline Noradrenaline

2.8 ~ 0.3 1.6 ~z 0.4 1.8 ± 0.1 1.7 ~ 0.2

11.7 ± 9.5 ± 4.9 ~ 4.5 ±

Tryptamine

9.6 ± 0.5

8.1 ± 0.3

Histamine Metaraminol

0.8 5_ 0.1 1.1 ± 0.1

0.2 ± 0.01 1.1 -L=0.3

0.7 0.6 0.3 0.6

(1) The uptake occurs against a considerable concentration gradient, since in the membrane vesicles a concentration more than 100 times higher than that of the medium builds upX4,23. (2) With increasing concentrations of noradrenaline in the incubation medium, the uptake of this amine reaches a saturation level 23. (3) The uptake of dopamine, noradrenaline and adrenaline is activated by ATP and Mg 2+ (Table I) 1,~1. (4) The stimulation of the uptake by Mg2+/ATP is limited to some amines, e.g. CA and 5-hydroxytryptamine (5-HT), whereas others, e.g. tryptamine, histamine and metaraminol, are not markedly influenced by Mg ~+ and the nucleotide (Table I) 14. Furthermore, the Mg2+/ATP-stimulated uptake of noradrenaline shows some stereospecificity, (--)noradrenaline being taken up preferentially 23. (5) The Mg2+/ATP-stimulated CA uptake markedly decreases with diminishing temperature, whereas the CA uptake in the absence of Mg2+/ATP as well as the uptake of tryptamine (which is considerable) are only slightly temperature-dependent 14 (Fig. 1). (6) N-Ethylmaleimide, an inhibitor of ATPase, as well as reserpine decrease the Mg2+/ATP-dependent CA uptake, but do not markedly influence the uptake of tryptaminO4, 24 (Table II). (7) A stoichiometric correlation between the ATPase activity of the vesicles and the influx of CA across the membrane has been demonstrated with various methods12,22,24. F r o m these and other findings it may be concluded that the transport of CA through the granular membrane does not occur by mere passive diffusion, but rather that a specific mechanism is involved. The connection of the transport with ATP and ATPase probably indicates its dependence on energy which enables the amine to be pumped into the vesicular space.

319

MECHANISMS OF CATECHOLAMINE ACCUMULATION

lntragranular interactions In monoamine-storing organelles, the amines are accumulated in very high concentration together with other constituents, especially nucleotides. Thus, in 5-HT organelles of rabbit blood platelets, the concentration of 5-HT has been estimated to be 1.1 M (20 ~o, w/v) and that of ATP 0.5 M (25 ~o)17. Adrenal chromaffin granules of cow contain about 0.6 M (10 ~ ) CA, 0.15 M (7.5 ~ ) A T P 13 and in addition soluble proteins, among them the chromogranins 18. These storage organelles could hardly be osmotically stable if the amines and nucleotides were present in monomolecular form. Therefore, a physicochemical interaction between the constituents of the organelles probably occurs. In the following, the type of interaction was studied by using physicochemical methods.

(1) Ultracentrifugation Analytical ultracentrifugation shows that in artificial solution (e.g. modified Tyrode), noradrenaline and ATP, in a molar ratio of 3.5 : 1 (corresponding approximately to the ratio CA : A T P in adrenal chromattin granules), form mixed aggregates. The average apparent molecular weight increases with decreasing temperature and rising concentration of the solutes. Small amounts of bivalent (but not of monovalent) cations promote aggregation, whereas larger concentrations of these ions cause disaggregation z (Fig. 2). Noradrenaline or ATP alone, in the absence of bivalent cations, do not aggregate to a marked extent. However, on addition of earth alkali ions to a solution of ATP, adenosine-5'-diphosphate or guanosine-5'-triphosphate (which also occur in chromaffin granules), the apparent average molecular weight markedly increases, and this rise is further enhanced by adding noradrenaline to a solution containing A T P and bivalent cationsS, 7. Dopamine and adrenaline also aggregate with ATP, the latter, however, to a lesser extent than noradrenaline. In contrast, other amines, like tryptamine, tyramine and histamine, do not show marked aggregation with nucleotidesZ, 4. Based on results obtained from sedimentation equilibrium and velocity experiments, it can be estimated that in aqueous solutions corresponding in their composition to the content of chromaffin granules, more than TABLE II EFFECT OF RESERPINE AND N-ETHYLMALEIMIDE

(NEM) ON

UPTAKE OF RADIOACTIVE AMINES BY ISOLATED

MEMBRANES OF BOVINE ADRENAL CHROMAFFIN GRANULES INCUBATED IN AN ARTIFICIAL MEDIUMz l AT 3 7 ° C FOR 15 MIN

Concentrations in the incubation medium: amines 45 /tM, ATP 5 raM, reserpine 8.2 × 10-6 M, NEM 2.5 × 10-4 M. The values represent averages with S.E. of 3-5 experiments and are indicated in nmoles/mg protein 14. 5-HT = 5-hydroxytryptamine; DA = dopamine.

Amine

Controls

Reserpine

NEM

5-HT + ATP DA + ATP Tryptamine

13.0 4- 0.6 9.8 ~ 0.4 9.1 i 0.3

3.1 -4- 0.4 1.9 4- 0.2 8.2 5z 0.2

5.4 4- 0.1 1.2 4- 0.2 7.8 4- 0.4

320

A. PLETSCHERet a/. 3

centrifugolforce 10.000 .E o 10- ~

Dopamlne +ATP .~ Tryptamine

~

s

Dopamine

=

' '/;IA

-~ 6000~u4000-

.c_

i

)

~ 8000-

I

~

60 I

3

; 2000-

/TP

O-

o

2's

3)°c

Incubation temperature

~

o

5 10 15 20 25 30 Temperoture. *C

212j3L~l~Is)61Tlsls~o3

Fractionnumber

Fig. I. Temperature dependence of [14C]amine uptake by isolated membranes of bovine adrenal chromaffin granules incubated in an artificial mediumzl for 15 min. Initial concentrations: amines 45 pM, ATP 5 mM. The points indicate averages of 2 experiments. The deviation of the two values is indicated by the vertical lines 14. Fig. 2. Temperature dependence of apparent average molecular weights of mixtures of noradrenaline (NA) and adenosine-5'-triphosphate (ATP), molar ratio 3.5:1, total concentration 17%, w/w 6. (1) No further addition; (2) addition of CaClz (molar ratio CaCI~/NA = 0.08), (3) molar ratio CaCI~/ NA = 0.25 (molar ratio NA/ATP reduced to 3); (4) addition of CaClz and MgC12(molar ratio CaCI2/ NA = 0.08, molar ratio MgC12/NA -- 2)6. Fig. 3. Content of chromaffin granules: concentrations of adrenaline (A), noradrenaline (NA) and adenosine-5'-triphosphate (ATP) in different fractions after centrifugation at 196,000 × g (tube bottom) and 1 °C for 35 h in a swinging bucket rotor. Original concentration of catecholamines ÷ ATP, 2.4% w/v. Fraction 10 corresponds to the bottom of the tube 9.

20 molecules of C A and A T P show mutual interaction at 37 °C, Preliminary measurements o f osmolality o f C A - A T P - C a C l z mixtures confirm the occurrence o f intermolecular bonding at 37 °C. On analytical ultracentrifugation o f a solution o f n o r a d r e n a l i n e - A T P - C a 2+ (molar ratio 4:1:0.25) to which 2-3 % chromogranins (obtained f r o m bovine adrenal chromaffin granules) are added, a single Schlieren peak appears at various temperatures and concentrations. The sedimentation velocity o f the observed Schlieren peak is higher than that obtained with solutions o f either n o r a d r e n a l i n e - A T P - C a 2+ or of the chromogranins alone. A l b u m i n does not sediment together with noradrenaline and A T P 8. These findings indicate that aggregates o f noradrenaline and A T P probably interact with the chromogranins. In the isolated contents o f adrenal chromaffin granules, too, noradrenaline, adrenaline and A T P sediment jointly 9 (Fig. 3). At the field of gravity used, the sedimentation rate is much higher than the rate to be expected if the solutes were present as single molecules. Furthermore, the average apparent molecular weight of the granular contents markedly increases with rising concentration and diminishing temperature, whereas the concentration and temperature dependence of the molecular weights o f the chromogranins are far less pronounced. These experiments indicate that CA and nucleotides such as A T P interact also in vivo in the chromatfin granules.

MECHANISMS OF CATECHOLAMINE ACCUMULATION

321

C

O tJ ¢. T = 1.6 nsec

8 O U.

1"< 0 . 4

n sec

Wavelength (nm) Fig. 4. Fluorescence spectra o f chromaffin granules disrupted in distilled water (top curve) and o f

intact granules of the same concentration in a solution of 10% saccharose and 0.9% NaC1 (lower curve), r means fluorescencelife time19.

(2) Fluorescence spectroscopy The experiments described show that CA in aqueous solutions as well as in the content of disrupted chromaffin granules interacts with nucleotides such as ATP. In order to confirm the interaction also under in vivo conditions, the quantum yield, the life time and the degree of polarization of the CA fluorescence o f intact isolated chromaffin granules were measured and compared to those o f artificial solutions of CA with and without ATP. In the CA storage granules as well as in artificial mixtures of CA plus nucleotides, the main fluorescence originates from the catechol group. With fluorescence spectroscopy, the environment of a fluorescent group can be investigated, and therefore evidence for a possible interaction of the CA with other molecules, e.g. nucleotide, may be obtained. (a) Fluorescence yields and life times. In solutions of CA and mixtures of CA plus ATP and bivalent metals, the fluorescence quantum yield decreases with rising concentration of the solutes (fluorescence quenching) and approaches zero at higher concentrations. Since the fluorescence life time diminishes with rising concentration, the quenching originates from collisions with the catechol group (dynamic quenching) and not from strong binding of the CA molecules among themselves or to ATP molecules (static quenching) 19. In fact, in the case of static quenching the life time should not change because the remaining fluorescence originates from the free CA left which behave like CA in dilute solutions.

A. PLErSCHER eta/.

322 0.300.25 0.20 0.15 0.10 C 0

~= 0.05 U~

0 O.

I

[

I

I

]

20

2'5

3'0

35

0.30 0.25 0.20-

,-,

0.150.10

0.050

0

5

10

1'5

Adrenaline concentration in % (w/v)

Fig. 5. Degree of fluorescence polarization (p) as a function of the concentration in artificial solutions. Lower curve: adrenaline + CaCI2, molar ratio 3.6:0.25; upper curve: adrenaline + ATP ÷ CaCI2, molar ratio 3.6:1:0.25. Excitation and fluorescence wave length 280 nm and 320 nm respectively; band width 10 nm 19.

The finding that the quenching is collision-induced indicates that the catechol groups, even in the presence of ATP, retain a relatively good mobility. However, quenching of the CA fluorescence is stronger and fluorescence life time shorter in the presence of ATP indicating that some interaction between CA and ATP must occur. This interaction may possibly be explained by binding of the positively charged nitrogen of adrenaline to the negatively charged phosphate group of ATP forcing the catechol groups into positions which would increase the probability for collision. In suspensions of whole chromaffin granules in which the CA are localized in high concentration within the granules, strong quenching and shortening of life time occurs as opposed to solutions of disrupted granules where the granular content is equally distributed in the aqueous medium 19 (Fig. 4). These observations indicate that the fluorescence quenching depends on collisions and that within the granules the degree of mobility of the CA is also relatively high. (b) Polarization of CA fluorescence. The P-value (degree of polarization of the lowest singlet-singlet transition) has been shown to be a measure for mutual interaction between CA and ATP in artificial solutions. Thus, in mixtures of CA and ATP

323

M E C H A N I S M S OF C A T E C H O L A M I N E A C C U M U L A T I O N T

16-

RAT

RABBIT

14o. 1 2 -

(17)

<~ 1 0 o

o ~

o

182~ 21(birth)

3r5

80

l

I

1820

I

28(birth)

, II

42

135

Days after conception Fig. 6. Molar ratios of catecholamines (CA)/adenosine-5'-triphosphate of adrenals of rats and rabbits on various days after conception. Each value represents a mean with S.E. Number of experiments in parentheses. All values except that for 20 days were significantly different from that at 18 days (P < 0.01).

in which aggregation occurs as measured by other means (e.g. ultracentrifugation 2, infrared 16 and nuclear magnetic resonance 2a spectroscopy), the P-value is higher than in solutions of the same concentration of CA alone 19 (Fig. 5). Furthermore, in noradrenaline-ATP mixtures, a biphasic change of the P-value as a function of the Mg 2÷ concentration takes place which parallels the Mg2+-induced biphasic alterations of the apparent average molecular weights 2 . The P-value measured in suspensions of whole chromaffin granules ranges between 0.18 and 0.22. Comparisons with artificial solutions (Fig. 5) indicate that P-values of this magnitude must result from an interaction of CA, e.g. with ATP, and cannot be due to the presence of CA alone in the concentrations found in the granules (10-15%, w/v). In fact, adrenaline, in concentrations of 10-15%, yields P-values of 0.11-0.15 only, whereas in mixtures of the same amounts of adrenaline and ATP (molar relation 3.6:1) the P-values were as high as 0.17-0.22 (Fig. 5). The polarization experiments thus confirm that in the intact chromaffin granules the catecholamines interact with ATP, whereby - - according to the quenching and life time measurements - - the catechol groups retain a relatively high degree of mobility. The present experiments do not, however, indicate to what extent the chromogranins, which have been shown to interact with C A - A T P aggregates in vitro 8, contribute to the relatively high P-value of the granules.

Ontogeny of monoamine-storing organelles The presence of nucleotides in the chromaffin granules is probably of importance for the storage of CA (see below). It was therefore of interest to investigate whether,

324

A. PLETSCHERel a/.

during ontogeny of the adrenal medulla, the storage of ATP preceded that of CA. Biochemical analysis of the adrenal medulla of rabbits and rats during pre- and postnatal development showed that the molar ratio of CA to ATP increased by a factor 20-60 from day 18 after conception to the adult age ~ (Fig. 6). Since the major part of CA and A T P of the adrenals is probably localized within the chromaffin granules of the medulla ~s, it can be assumed that in the earliest stages of development these granules contain primarily ATP, whereas the storage of higher amounts of CA develops only later. The biochemical results are in agreement with electron microscopic findings in rats it. Accordingly, the fetal adrenal medulla shows numerous emptylooking organelles (ATP does not give an osmiophilic reaction under the conditions of the experiment) long before CA are biochemically detectable. In the course of pre- and postnatal development, an increasing number of vesicles exhibits a dense osmiophilic core probably due to the storage of CA. These findings with adrenal medulla are corroborated by those with megakaryocytes (stem cells of blood platelets) and with blood platelets which are able to store 5-HT, an amine also showing aggregation with ATP ~. By combined electron microscopic and biochemical techniques, evidence has been presented that the megakaryocytes contain at best small amounts Meier ratio CA:ATP:Cd'=4:I:0.2 G

availob(e for

membrane

permeation

C,A---A~p---C~ 2T~--CA. \

//

/ /

/

~

i

i ~

CA Af'P

,,~'%

\

"CA 6 C,A

\

~e',. ~,--ATP---CA~,...AT'P ,ca c# C~ CA cA \ '

"

'C

" ~

,

A""~CA~c~-"A~

I r'~* J CJ~ dA I

,"-~ ",

'~'CA Ca,

"/A~P---C~.~ . . . . .

/ ~,! '- ~

~ ) ' ~" ," ~ C.~_.ATp CA ~ --%.,~

i

c'~ ~ A @ I Ca-- ATIa-~2A , \ "'-ATI~ | /

CA. ' , C:^

CA ~ a

/

~.~7~---c~ 'Ar~--~c,~ ~,Rre.' c~

bA ~a ~..J,,"

/ ".

i

\ 7/"cA c,A ~:~.. ,,,

\

I

,

' ""C

"t..,,I1,.

/

, J

/

Q-A~r

"

._Ca A~k__CAk~)CA,,

i

/

',

',~fp /

\ \ &~~'k". '7':.o_..-"%-ca .CA ..~, .:A>j ' ~ A / ,.A,~' t-k "C~' ..~.~ " ' " ' C 6 ' "b.~ \ ca ~ C ® ~',~ "';AT~'~,~ "~A ,c~ / C,¢ "" ', "'CA'" \ cA..o,TmA'," cA

~"'ZTp---CA

k

~

"' ~ ' "

ATP---Ca. " ~"'

) /

"'- "

/

C£ /

-

---fiuctuo.tl,,g

-

Londs

Membrane pump

Fig. 7. Model representing the catecholamine (CA) storage in chromaffin granules. The interaction between CA, ATP and bivalent cations is of a dynamic nature. In addition, an interaction with chromogranins has been shown to occur.

MECHANISMS OF CATECHOLAMINEACCUMULATION

325

of 5-HT, but that numerous empty-looking organelles, probably storing ATP, are present in these cells. Furthermore, in platelets of guinea pigs, 'empty' organelles storing high amounts of ATP, but little 5-HT, have been demonstrated and isolated. On exposure of the megakaryocytes or platelets to 5-HT (e.g. by injection of the amine), these organelles become highly osmiophilic due to accumulation of the aminel0, 25. Therefore, the presently available evidence indicates that the storage of ATP precedes that of CA in the adrenal medulla.

Summary and conclusions According to the above findings, two different processes seem to be involved in the storage of CA in adrenal chromaffin granules. The amines are apparently pumped into the organelles by an Mg~+/ATP-dependent active mechanism operating at the level of the granular membrane and involving ATPase. This mechanism alone is hardly responsible for the very high content of CA normally present in chromaffin granules. Previous experiments have shown that the maximum noradrenaline concentration reached in isolated granular membranes as a consequence of an active transport amounts to 5 mM (see ref. 1). However, the CA concentration in intact chromaffin granules was calculated to be approximately 500 mM. The build-up of this high concentration is most likely due to the existence of the second mechanism, i.e. the intragranular interaction of the CA with nucleotides such as ATP (which probably preexists in the granules before the amines are stored) and also with chromogranins. This interaction probably reduces the diffusion of CA out of the granules enabling the amine pump to operate against a much higher amine concentration gradient. Preliminary experiments with artificial lipid membranes confirm this view, since addition of ATP and CaCI~ to noradrenaline or adrenaline solutions decreases the velocity of membrane permeation of the amines probably as a result of a reduction of the concentration of non-aggregated amine. On the other hand, the binding of CA in the organelles is of a dynamic nature and rather loose as indicated by the fluorescence measurements. This reversibility of the storage process allows the amines to be rapidly liberated, e.g. during the release process of CA. The two mechanisms are summarized in Fig. 7 which is a hypothetical model demonstrating the mode of storage of catecholamines in adrenal chromattin granules.

1 AGOSTINI,B., AND TAUGNER,G., The membrane of the catecholamine storage vesicles of the adrenal medulla. Correlation of ultrastructure with biochemical properties, Histochemie, 33 (1973) 255-272. 2 BERNEIS, K. H., PLETSCHER,A., AND DA PRADA, M., Metal-dependent aggregation of biogenic amines: a hypothesis for their storage and release, Nature (Lond.), 244 (1969) 281-283. 3 BERNEIS,K. H., DA PRADA, M., AND PLETSCHER,A., Micelle formation between 5-hydroxytryptamine and adenosine triphosphate in platelet storage organelles, Science, 165 (1969) 913-914. 4 BERNEIS,K. H., DA PRADA, M., AND PLETSCHER,A., Physicochemical properties of 5-hydroxytryptamine organelles of blood platelets, Agents Actions, 1 (1969) 35-38. 5 BERNEIS,K. H., DA PRADA,M., AND PLETSCHER,A., Metal-dependent aggregation of nucleotides with formation of biphasic liquid systems, Biochim. biophys. Acta (Amst.), 215 (1970) 547-549. 6 BERNEIS,K. H., PLETSCHER,A., AND DA PRADA,M., Phase separation in solutions of noradren-

326

A. PLETSCHER e t a [ .

aline and adenosine triphosphate: influence of bivalent cations and drugs, Brit. J. Pharmacol., 39 (1970) 382-389. 7 BERNEIS, K. H., DA PRADA, M., AND PLETSCHER,A., A possible mechanism for the uptake of biogenic monoamines by storage organelles: incorporation into nucleotide-metal aggregates, Experientia (Basel), 27 (1971) 917-918. 8 BERNEIS,K. H., GOETZ, U., DA PRADA, M., AND PLETSCHER,A., Interaction of aggregated catecholamines and nucleotides with intragranular proteins, Naunyn-Schmiedeberg's Arch. exp. Path. Pharmak., 277 (1973) 291-296. 9 DA PRADA, M., BERNEIS,K. H., AND PLETSCHER,A., Storage of catecholamines in adrenal medullary granules: formation of aggregates with nucleotides, Life Sci., 10 (1971) 639-646. 10 DA PRADA, M., PLETSCHER,A., AND TRANZER,J, P., Storage of ATP and 5-hydroxytryptamine in blood platelets of guinea pigs, J. Physiol. (Lond.), 217 (1971) 679-688. 11 ELFVIN,L. G., The development of the secretory granules in the rat adrenal medulla, J. Ultrastruct. Res., 17 (1967) 45-62. 12 HASSELBACH,W., AND TAUGNER,G., The effect of a cross bridging thiol reagent on the catecholamine fluxes of adrenal medulla vesicles, Biochem. J., 119 (1970) 265-271. 13 HILLARP, N.-A., Adenosinephosphates and inorganic phosphate in the adrenaline and noradrenaline containing granules of the adrenal medulla, Acta physiol, scand., 42 (1958) 321-332. 14 LOTOLD,B., DA PRADA, M., AND PLETSCHER,A., in preparation. 15 O'BRIEN, R. A., DA PRADA, M., AND PLETSCHER, A., The ontogenesis of catecholamine and adenosine-5'-triphosphate in the adrenal medulla, Life Sci., 11 (1972) 749-759. 16 PAI, V. S., AND MAYNERT,E. W., Interactions of catecholamines with adenosine triphosphate in solutions and adrenal medullary granules, Molec. Pharmacol., 8 (1972) 82-87. 17 PLETSCHER,A., DA PRADA,M., ANDTRANZER,J. P., Transfer and storage of biogenic monoamines in subcellular organeiles of blood platelets. In K. AKERTAND P. G. WASER(Eds.), Mechanisms t~f Synaptic Transmission, Progress in Brain Research, Vol. 31, Elsevier, Amsterdam, 1969, pp. 47-52. 18 SMtTH,A. D., Biochemistry of adrenal chromaffin granules. In P. N. CAMPBELL(Ed.), 7he Interaction of Drugs and Subcellular Components in Animal Cells, Churchill, London, 1968, pp. 239-292. 19 STEFFEN,H., DA PRADA, M., AND PLETSCHER,A., Fluorescence properties of catecholamines in isolated storage organelles of adrenal medulla, Submitted for publication. 20 STJ)~RNE,L., The synthesis, uptake and storage of catecholamines in the adrenal medulla. The effect of drugs. In H. BLASCHKOAND E. MUSCHOLL(Eds.), Catecholamines, Springer, Berlin, 1972, pp. 231-269. 21 TAUGNER,G., The membrane of catecholamine storage vesicles of adrenal medulla. Catecholamine fluxes and ATPase activity, Naunyn-Schmiedeberg's Arch. exp. Path. Pharmak., 270 (1971) 392-406. 22 TAUGNER,G., The effects of salts on catecholamine fluxes and adenosine triphosphatase activity in storage vesicles from adrenal medulla, Biochem. J., 123 (1971) 219-225. 23 TAUGNER,G., The membrane of eatechotamine storage vesicles of adrenal medulla. Uptake and release of noradrenaline in relation to the pH and the concentration and steric configuration of the amine present in the medium, Naunyn-Schmiedeberg's Arch. exp. Path. Pharmak., 274 (1972) 299-314. 24 TAUGNER,G., UND HASSELBACH,W., Die Bedeutung der Sulfhydryl-Gruppen for den Catecholamin-Transport der Vesikel des Nebennierenmarkes, Naunyn-Schmiedeberg's Arch. exp. Path. Pharmak., 260 (1968) 58-79. 25 TRANZER,J. P., DA PRADA, M., AND PLETSCHER,A., Storage of 5-hydroxytryptamine in megakaryocytes, J. Cell Biol., 52 (1972) 191-197. 26 WEINER, N., AND JARDETZKY,O., A study of catecholamine nucleotide complexes by nuclear magnetic resonance spectroscopy, Naunyn-Schmiedeberg 's Arch. exp. Path. Pharmak., 248 (1964) 308-318.