Acta physiol. scand. 1976.96. 512-525 From the Department of Pharmacology, Karolinska Institutet, Stockholm, Sweden

An in Vitro-Formed Protamine-Heparin Complex as a Model for a Two-Compartment Store for Biogenic Amines BY BORJEUVNASand CARL-HUGO ABORG Received 20 August 1975

Abstract UVNAS,B. and C.-H. ABORG.An in uitro-formedprotamine-heparin complex as a model for a two-compartment store for biogenic amines. Acta physiol. scand. 1976. 96. 512-525. The capacity of an in vitro-formed protamine-heparin complex (PHC) to store inorganic cations and biogenic amines was investigated. The PHC behaves like a two-compartment storage system. One compartment corresponds to the terminal free carboxyl groups of the protamine moiety and has the characteristics of a cation exchanger, with the ability to bind inorganic cations and biogenic amines in a reversible and rather unselective manner. The cations and biogenic amines therefore compete for and displace each other from the common ionic binding sites. The binding sites in the other compartment, corresponding mainly to the carboxyl groups of the heparin moiety, are only unmasked at high ionic concentrations and show a specific affinity for biogenic amines. The storage of amines in this compartment of the PHC is reversible but is dependent not only o n simple ionic binding but evidently also on other attractive forces, such as dipole and hydrogen bonding.

In a previous paper (Aborg and Uvnas 1968) we reported on the cation-binding properties of a protamine-heparin complex (PHC) formed in uitro. The binding of inorganic cations and biogenic amines to this complex showed a pH-dependence indicative of an ionic linkage to carboxyl groups. The storage capacity of the complex corresponded to the number of free terminal carboxyl groups in the protamine part of the PHC. We have also demonstrated that the matrix of the basophil granules of rat peritoneal mast cells consists essentially of a complex formed between a basic, low molecular weight polypeptide and heparin (Bergqvist, Samuelsson and Uvnas 1971). In uitro the cation-storing properties of the mast cell granules are qualitatively similar to those of the PHC (Uvnas and Thon 1966). Further studies revealed the presence in adrenal medullary granules (Fillion, NosPl and Uvnas 1971) and in adrenergic vesicle preparations from various organs and tissues (Aborg et al. 1972) of a mucopolysaccharide-containing material capable of binding inorganic cations and biogenic amines reversibly. However, the amounts of amines taken up by such material in uitro were considerably smaller than those known to be stored in various adrenergic depots in vivo. It is well known that the amounts of amines stored in uivo are in fact SO 512

A MODEL FOR BlOGENlC AMlNE STORE

513

large that they must be assumed to be present in very high concentrations in the storing structures. If one assumes that the amines are stored in solution, then the amine concentrations in adrenal medullary or nerve terminal granules can be calculated to be around 0.5 M (Green 1962). Since in our previous in uitro uptake and release studies (see above) we used amine concentrations which were considerably lower than this, we decided to carry out a detailed reinvestigation of the storage properties of our various mucopolysaccharide-containing materials using amine concentrations close to those assumed to occur in the aminestoring vesicles in viuo. The present investigation is an extension of our earlier study on the cation storing properties of the PHC. This complex was found to behave like a “two-pool” biogenic amine store. In agreement with our previous report, the smaller “pool”, localized to the protamine terminal carboxyl groups, exhibited the properties of a cation exchanger, with a rather unselective affinity for both inorganic cations and biogenic amines. The second (and larger) “pool”, which corresponded to the carboxyl groups (and probably only to a minor extent the sulphate groups) of heparin had a more specific affinity for biogenic amines. These findings may have general implications for the understanding of the mechanisms of storage and release of biogenic amines and other electrically-charged compounds.

Methods The PHC was prepared by mixing solutions of protamine sulphate (0.5 g) and sodium heparinate (0.8 g) in 0.9% NaCl solution at pH 7 whilst stirring vigorously (Aborg and Uvnas 1968). The milky precipitate formed was centrifuged down at 3 000 g for 30 min, washed with 4 x 25 ml of deionized water and then suspended in 7 ml of deionized water and stored at -1-4°C.The content of dry material (dried in uacuo over P,O, for 3 days) in the suspension varied somewhat (46.449.4 mg/ml) between the various preparations. Profamine base was prepared by running a solution of protamine sulphate in deionized water through a column of Dowex 2-X8 ion-exchanger (20-50 mesh) as previously described (Aborg and Uvnas 1968). After elution with water, the material was lyophilized and stored in uacuo over PaO,. Uptake studies Plastic tubes with stoppers were carefully weighed. To each tube was added 2 ml of freshly-prepared sodium phosphate buffer (pH 7) with an appropriate admixturc of ,,NaCI or 2 ml of 5-6:/, H,SO, solution, p H 6, containing one of the “C-labelled amines, followed by 0.1 ml of PHC suspension. After 10 min incubation, the samples were centrifuged for 20 min at 3 000 x g . The clear supernatants were sucked off, except for 1-2 drops which were left in the tubes in order to minimize the loss of PHC. Each tube was reweighed to allow the amount of radioactivity in the remaining supernatant to be calculated, thereby enabling a reliable estimate of the uptake of sodium and amines by the PHC to be made. After drying the PHC was suspended in 300 ,ul of 0.4 N HCI, heated at 60°C for 10 min and then centrifuged a t 3 000 xg for 20 min. 100 pI of the supernatant was used for scintillation counting. When calculating the uptake, corrections were made for changes in the concentration of the amine or sodium in the suspension fluid due to uptake by the PHC. Uptake of sodium: low conceniration (phuse I uptake) 100 ml of 0.1 N NaOH was titrated to pH 7.35 with conc. H,P04 (Agla micrometer syringe) and diluted with deionized water to give incubation fluids containing 100 (stock solution) 30, 10, 3, 1, 0.3, 0.1 and 0.03 peq Na+/ml. To 9 ml of incubation fluid was added 50 pI of carrier-free ,,NaCI (50 pCi/ml). 0.1 ml of

PHC suspension was incubated in 2 ml of the resulting fluid. Uptake of sodium: high concentration (phase I1 uptake) About 2 gm of NaOH was dissolved in deionized water, titrated with 5-6% H,SO, to pH 7 and diluted t o 100 ml with deionized water. The resulting solution-about 0.5 N-was diluted with deionized water to

33 - 765874

514

BORJE UVNAS AND CARL-HUGO

KBORG

TABLE I. Titration of the prolamine base with 5 M HCI in deionized water and 1 M KCI respectively. Protamine base

Groups titrated

Peq

Molar ratio

Molecular weight

511.4 mg in I M KCI

Guanidino weaker Amino or imino Carboxyls Guanidino weaker Amino or imino Carboxyls

2 190 222 145 1890 190 135

30 3 2 28 3 (2.8) 2

7 416 7 316 7 468 7 018 7479 7 018 7 286

473.7 mg in deionized water

Mean value

HCI

yield incubation fluids containing about 500 (stock solution), 400, 300, 200, 100, 30 and 10 peq Na+/ml. To 5 ml of incubation fluid was added 20 pI of carrier-free erNaCl (50 pCi/ml). 0. I ml of P H C suspension was incubated in 2 ml of the resulting fluid. Uptake of sodium: influence of p H

About 200 mg of H,BO, and 0.05 ml of carrier-free 22NaCI (50 pCi/ml) were added to 100 ml of freshlyprepared 0.01 N NaOH. The solution was acidified stepwise t o about pH 7.5 by adding (Agla micrometer syringe) conc. H,PO, and then acidificed t o p H 3 with concentrated citric acid. In other experiments, 0.01 N NaOH was acidified t o about p H 7 with conc. H,PO, and then acidified to pH 4.5 and incubated with 0.1 ml of the PHC suspension in the presence of appropriate amounts of 2eNaCI(see above). The nature of the anion did not influence the pH-dependence of the sodium uptake. Uptake nf phenylethylamine and adrenaline: phase I and I I uptakes Solutions of the above amines were titrated to pH 6 with 5-6':; H,SO, and diluted with deionized water to give the concentrations desired, as described above for sodium phase I and I 1 uptakes. H,SO, was used since i t yields water-soluble salts (PhEA), prevents oxidation (A) and gives a pH of around 6-7. Considering these advantages, the drawback of the rather poor buffering capacity of sulphite was knowingly accepted. Proposed gross structure of the PHC Protamines are characterized a s low molecular weight, straight-chain polypeptides, with arginine as the main basic amino acid. The polypeptide is believed t o have only two free groups at pH 7-a carboxyl group a t one end and an amino (or imino) group at the other end of the open peptide chain (Linderstrom-Lang 1935). Unfortunately, no data were available concerning the homogeneity and amino acid composition of the prolamine we used. We therefore tried to obtain the necessary information about the gross structure of our PHC by titration. Two samples of prolamine base (473.7 and 541.4 mg) obtained from protamine sulphate (see p. 513). were titrated with 5 N HCI in deionized water and I M KCI respectively. According t o Linderstrom-Lang (1935), the consumption of HCI above p H 10 corresponds to guanidino groups, between pHs 10 and 5 to weaker amino and imino groups and between pHs 5 and 2 to carboxyl groups. Our titration results (Table I ) indicate a ratio between guanidino, amino (imino) and carboxyl groups of 30:3:2. From the titration results, the minimum molecular weight was estimated to be 7 286 (7 018-7 479, n = 6): in the calculations below we have used a value of 7 300. Using the microbiuret technique with prolamine base as reference, 56':: of our PHC was found t o be protamine. Previous titration of heparinic acid (Heparin, Vitrum") revealed about 44% acid groups per mole (actual titration value: 43.6) (Aborg and Uvnas 1.c.). The heparin used is claimed to have 1 carboxyl and 2 sulphate groups per disaccharide unit. Since 1 mol of protamine base contains about 30 guanidino groups, we may assume our complex t o be composed of 2 mol of heparinic acid and 3 mol of prolamine base-providing that the ionic bonds between the negatively-charged groups of heparin and the positively-charged guanidino groups of protamine occur as depicted in Fig. I . The molecular weight of the PHC would then be approximately 38 000 (2 x 8 OOOi 3 x 7 300). It should be emphasized that we have not attempted to carry out a detailed analysis of the structure of our PHC. Our aim has only been t o obtain data which were sufficiently informative t o illustrate the principles behind the ability of the P H C to store inorganic cations and biogenic amines.

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A MODEL FOR BIOGENIC AMINE STORE

Materials Heparin (mucous), activity 137.8 U./mg dried material: AB Vitrum, Stockholm. Protamine sulphate (from herring): Sigma Grade Ill, essentially histone-free; DL-adrenaline (anhydrous) and j3-phenylethyIamine (free base): Sigma Chemical Co., St. Louis, Mo., USA. DL-Adrenaline (~arbinol-'~C)DL-bitartrate and **NaCl( 3 - 100 mCi/mg): The Radiochemical Centre, Amersham, England. j3-Phenylethylamine I-14C. HCI: New England Nuclear Chemicals GmbH Frankfurt/M., West Germany. Abbreuiutions: A = Adrenaline; PHC= Protamine-heparin complex; PhEA = Phenylethylamine.

Results Biphasic uptake of sodium, adrenaline and phenylethylamine at p H 7 Sodium

As previously reported (Aborg and Uvnas I.c.), the PHC took up sodium ions when suspended in sodium phosphate buffer. The uptake increased with increasing buffer concentrations to level off at 80 neq/mg of PHC when the sodium concentration in the suspension medium approached 10 peq/ml. However, the fact that there is a further increase in the uptake of sodium at higher buffer concentrations has not been reported earlier. The uptake rose steeply when the sodium concentration approached 300-500 ,ueq/ml (Fig. 2). Adrenaline

Adrenaline (A) showed an even more pronounced biphasic uptake than did sodium (Fig. 3). The first phase of the uptake could be seen to level off at about 80 neq/mg PHC, as was the

I 200

0.01 0.03 0.1

0.3

I

3

10

30

100

n y

500

peq N a * / n l

Fig. 2. Biphasic uptake of sodium by the PHC. 0-0, Experimentally observed total uptake; 0-0, Phase I uptake calculated according to the Rothmund-Kornfeld equation (Fig. 7), pH 7.4 U, 83 neq/mg; x - x , Phase II uptake obtained by deducting phase I uptake from the total uptake. Incubation pHs (7.09-7.33) are indicated on the figure.

516

B ~ R J EUVNAS AND CARL-HUGO

ABORG

ioo I

500

-500

100

400

E" 100

300

:

0

2

E

!OO

200

100

100

' Y

001 003

0.1

03

I

3

pep A l m l

Fig. 3

10

30

100

500

0.03 0.1

Q3

I 3 10 psq PhEA I m l

Jo

100

500

Fig. 4

Fig. 3. Biphasic uptake of adrenaline by the PHC. 0-0, Experimentally obtained values; 0-0, Phase I uptake calculated according to the Rothmund-Kornfeld equation (Fig. 7),pH 7.4 U,,83 neq/mg; x - , Phase I1 uptake (obtained by deducting the phase I uptake from the total uptake). Incubation pHs (6.15-6.87) are denoted on the figure. Points no. 2, 6 and 8 were obtained by lowering the A conc. in the suspension medium from 500 meg/ml t o 400, 200 and 100 respectively. Note the reversibility of the A storage in the PHC and the rather good agreement with the corresponding primary uptake values. Fig. 4. Biphasic uptake of phenylethylamine by the PHC. 0-0, Experimentally observed total uptake; 0-0, Phase I uptake calculated according to the Rothmund-Kornfeld equation (Fig. 7). p H 7.4. U, 83 neqlmg; x --x, Phase I1 uptake (obtained by deducting the phase I uptake from the total uptake). Incubation pHs (7.09-7.33) are denoted on the figure.

case with sodium. This was clearly seen when the theoretical uptake at p H 7.4 was calculated (see below). However, the uptake again rose steeply when the PHC was exposed to A concentrations exceeding 10 peqlml. The highest uptake observed was 560 neq/mg PHC (at an A concentration of 0.5 M). Phenylethylamine

The uptake curve for PhEA was biphasic and very similar to that of A (Fig. 4). In the following discussion the two sections of the biphasic uptake curve will be referred to as phase I uptake and phase I1 uptake respectively. Comments As will be discussed below, the theoretical maximal uptakes of sodium, A and PhEA were

considerably higher than those actually recorded. These maximal uptakes might never be reached due to dissolution of the PHC at the high concentrations of sodium or amines required to achieve them.

A MODEL FOR BIOGENIC AMINE STORE

517

200

I50 V

I

a

r

\

100

4 C

50

4

5

6

7

8

9

10

1 I

Fig. 5. Influence of pH on the uptake of sodium (9.5peqlml) and adrenaline (0.95 peq/ ml). Extrapolated curves (O---O). Exp. observed uptake of sodium (0-0) and of adrenaline(0-0). Uptakeofsodium( -x ) and of adrenaline (A-A) calculated from Fig. 2 and 3 respectively according to the Rothmund-Kornfeld equation (Fig. 7).

1213

PH

Phase I uptake Influence of sodium and amine concentrations

The uptake curves for Na, A and PhEA tended to level off at around 80 neq/mg (Fig. 2-4). As described below, the uptake was very pH-dependent and even small reductions in the pH of the supensions medium below neutrality resulted in a noticeable reduction in uptake. This was especially evident in the A and PhEA uptake curves since, due to the poor buffering capacity of the sulphite, the pH fell, especially at lower amine concentrations, to pH 6.5 and below. However, after correction for these pH deviations according to the RothmundKornfeld equation the uptake curves for Na, A and PhEA showed very similar courses (Fig. 2-4). Influence of p H

The curve showing the sodium uptake at various pHs (Fig. 5 ) was biphasic with an inflexion around pH 7 and a sodium uptake at this pH of around 80 neq/mg PHC. The sodium binding ability decreased rapidly as the pH was reduced and was virtually abolished at pH 4. Above pH 7 the uptake of sodium increased steeply to reach a new maximum (-210 neq/mg of PHC) at pH 12-13. The uptake curves for A (Fig. 5) and PhEA showed that the uptake of these substances was as dependent on pH as the uptake of sodium was. Comments

The similarities in the uptake curves and pH-dependency of the uptake of sodium, A and PhEA indicate that they are bound to identical sites. At least on the acid side of neutrality, the titration curves are similar to the dissociation curve for protein carboxyl groups. The uptakes of sodium, A and PhEA at pH 7.0 (80 neq/mg PHC) are consistent with the number of carboxyl groups in the PHC as depicted in Fig. 1. Assuming a mol.wt. of 38 OOO, three

518

BORJE UVNAS AND CARL-HUGO

ABORG

Fig. 6. Influence of concentration and pH on the uptake of sodium and adrenaline by the PHC. conc. sodium ( x - x); conc. A ( - - - -