Improvements in the isolation of noradrenaline storage vesicles from bovine splenic nerves

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I' pp' gs9-850, 1970 .

Pergamon Press

IMPROVEMENTS IN THE ISOLATION OF NOR.ADRENALINE STORAGE VESICLES FROM BOVINE SPLENIC NERVES z) H. Lagercrants, R. L, HIein and L. Stjärne Department of Physiology I, Karoliaska Institutet, Stockholm, Sweden (Received in final form 29 March 1970) Some success has been achieved in the isolation of NA vesicles from bovine splenic nerve trunk in this (1-3) and other laboratories (3-6) . Despite differences in methods, the beat NA vesicle fractions contained 0 . 5-0 . 8 wg NA~mg protein, The NA vesicle preparation most often used by others in this laboratory consisted of the 9 000 g supernatant (isotonic potassium phosphate buffer) produced after Ultra Turraz homogeniration or aqueesing nerve trunks betweea rollers . It was felt that further improvements could be made in this preparation by removal of microsomal and other low density contaminants . The rationale for attempting to produce greater purification of nerve trunk vesicles is to establish a firmer basis on which to compare these with adrenal medullary granules and eventually with nerve terminal vesicles . At present many hypotheses concerning sympathetic nerve physiology are still based on data from adrenal medullary granules (7), which can be isolated in relatively pure fractions (S) . There is considerable question about the justification for such extrapolation, Abb reviations _ass = indicates eaayme Û6P = glucose-6_phoephate ATP = adenosine triphosphate MAO = monoamine ozidase ß-GP = ß-glycerophosphate NA = noradrenaline CyOx = cytochrome c oxidase RSA = relative specific activity ED TA _- ethylenedinitrilo SA =specific activity tetraacetic acid SN = 10 500 g-10 min supernatant FI, FII, FIII = fractions I, II, TPP = thiamine pyrophosphate x) Permanent address : Dept . of Pharmacology, Univ . Mise . Med. Center, ac son, s. , U: S. A, 839

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This communication describes a method which effectively removes microsamal coats++++  =tioa from NA vesicles prepared from bovine spleaic serve truck by the use of sucrose-D2 0 gradients . Estimates of purification are based am the ability to achieve increased sedimeatable NA: protein ratios relative to the original homogenate an$ to those values previously reported by others . ßuaatitative activities of kaowa marker eaaymea for the primary subceLlular compoaeats are gives. Methods Spleaic serves , Bovine spleaic serves were obtained 20-30 min post mortem and immediately chilled with ice . Usually 10-15 g nerves were dissected, desheathed and minced with scissors, all in the cold. The mince was homogenized

(ww,

1:2, 5 or less) in 0. 25 M sucrose containing 1 mM tris-HCl buffer, pH

7.4-T. 6. Ia most experiments the homogenization was carried out in as Ultra Turraz apparatus (Janke & Kuakel, Freiburg) at a setting of 75 (Powerstat, Bristol Electric Co, Bristol, Coan. ) for 2, 5 mfg (low speed) . 1n some eaperimeats a setting of 150_200 for 30 sec (high speed) was used. In two experiments, the asrves were homogenized in a motor driven teflon pestle homogenicer (Y'ioma~t,aiass B) using 20 strokes at highest speed. la two eaperimeats the serves were squeeaed between teflan rollers . Homogenates were centrifuged at 600 gmax 10 min is a Servall refrigerated centrifuge : The supernatant was filtered through gauge to remove floating lipdd material. the filtrate was centrifuged 10 500 gmaz 10 min, and the SN was layered oa 4 density gradients . Density gradients . Two types of sucrose density gradients were made. 7a most ezperimeats sucrose was dissolved is D20 (Norwegian Hydro, 99. $ %). 1a some ezperimeats sucrose-H2 0 solutions were used simultaneously with those costaiaiag heavy water for comparisam purposes . Linear gradients were made raagiag from 0. 25 to 1. 5 M sucrose. Oae part of 0, 25 M sucrose, ei ther is 50 % H2O-50 °~o D20 or is pure H2O, was mized with 2 parts of 1. 5 M sucrose-D20 or 1.5 M sucrose-H20, respectively. The gradients with 4-5 ml

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SN oa top were centrifuged 100 000 g - 90 min is a Beckman Ultracentrifuge (h2 65B) using a SW 40 awiagiag bucket rotor . Gradient fractions were removed from beneath with hooked pasteur pipettes, diluted with the buffered 0.25 M sucrose and centrifuged is a Ti 50 rotor at 140 000 g -30 min, ïa some ezperiments the NA fractions were diluted with 1. 5 M suaroae -D 20 and mined with light sucrose aolulßoa to make new gradients . These were then receatrifuged is the swingout bucket rotor and are referred to as repuri$ed fractions. The pellets from fractions were reeuspended is the desired medium, usually 0.25 M sucrose, with the aid of a perepez rod sad vibration. Sediments from samples of the original SN and the 10500 g pellet were washed and resuspeaded similarly. NA assay was done fluoraanetrically (9) . C~Oz as as indicator of mitochoadrisl contamination was measured polarographically at 30°C using a Clark ozygea electrode. The reaction mizture contained 0.1 mM cytochrome c, 16 mM aacorbate and 75 mM phosphate buffer at pH 7. 5 is a total of 3 . 0 ml aqueous solul3oa. MAO as as indicator of mitochoadrial ccataxnination was measured radiometrically (10) . Several phosphate hydrolysing easymee were used ss markers, and re leseed inorganic phosphate was measured (ll). All reactions were is 3. 0 ml at 37-38° C for 30 or 60 min after as initial equilibration for 10 min. G 6 Pase was measured sa an indicator of microsomal contamination. The medium contained 40 mM tris-maleate-NaOH buffer at pH 6. 5, 2 mM EDTA (tris salt) and 10 mM G6P. Substrate concentration was sear optimal. P-G Pase (acid phosphatase) was measured as an indicator of lysosomal contaminatio~ . The medium contained 40 mM acetate buffer at pH 5. 0, 2 mM EDTA (trio salt) and 10 mM ~-GP . Substrate concentration was near optimal. TP Pase was measured as as indicator of contamination by Golgi membrane sad derivates (neurotubules ? ) (12) . The medium contained 40 mM trio-HC1 buffer at pH 7. 2, 5 mM MgC12 and 5 mM TPP.

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Adenosine triphosphatase was measured in order to determine if activity was present in our purest NA fractions . The basic medium contained 20 mM Tris_ HCl buffer at pH T.2, 100 mM KCl, 10 mM NaCl and 5 mM Mg-ATP. Results Homo~~aisatloa .

The evaluation of the various procedures (ree Methods) is

as follaavs : 1) f$gh speed Ultra Turraz caused tmaecessary breakage of both NA vesicles sad mitochondria.

2) Squeesiag between nylon rollers was as

improvement over (1), but saEfere from relatively low yields sad a require_ meat of relatively high medium: tissue ratios. Although this method gives a much reduced sediment at 10500 g, subsequent layering of the SN an sucrose_ D20 gradients was not found to give better yields in terms of NA ; protein ratio, nor were contaminating easymes reduced compared to (4) .

3) Hamoge-

aisatioa with a Potter Elvehjem tube sad tefloa pestle was less efflcieat fn disrupting the minced nerve to release NA vesicles . It did not reduce coatamiaatlng ensymes compared to (4) . 4) Law speed Ultra Turrsz homogenisation gave the best NA : protein rsttos in purified eedimeatable fractions with the lowest coâtsmiaatioa by subcellulsr components . Differential Ceatrifugatioa. Minced serve trunks were homogenised by low speed Ultra Turraz is eiüier 0:25 M sucrose_1 mM tris-HCl at pH T.4 or is 0.130 M potassium phosphate buffer at pH T.2 . After removal of a crude fraction (600 g-10 min), the filtered $N was subjected to a eerier of centrifugal forces . The distributions of NA, CyOx and protein were measured in each sediment in order to determine that speed at which the most mitochondria were removed with the minimum lose of NA vesicles from the SN (Fig. 1) . The optimum force was at 10 500 g -10 min . Similar results were obtained using either medium. Sucrose-DSO vs Sucrose-HZO Gradients .

Because co++~+~+~+f*,=tiom with frag-

meats of eadoplasmic reticulum appeared to be the major problem in the purification a£ NA vesicles from heart muscle (14,15), much of our initial interest was concerned with removing G 6 Pase activity from the NA fractions .

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843

FIG. 1. Centrifugation force (gx10 min) for optimal NA recovery with minimal mitochondrial contamination. CyOx and NA = % content.

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More than 90 % of thin enzyme is present in the 10 500 g SN . The appearance of sucrose-Fî20 and aucroae-D2 0 gradients is illustrated in Figa . 2 sad 3, respectively . FI occurred relatively concentrated in a band near the interface between SN and gradient . A thin line was usually obvious, below which occurred two white cloudy layers, FII and FIII . 1n, sucrose-D20 gradieata, there was usually a definite thin clear apace separating FII from FIII. In sucrose-Fî 20, this separation was not as sharp and more often coasisted of a broarder lean cloudy zone (Fig . 2) . If the NA containing FII and FIII were repurified in a second gradient (nee Methods), the same band appearance was fpuad with FI nearly absent . NA sad G 6Paee activity are plotted is Figa . 2 and 3 according to de Duve (16) with the RSA (eg .

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NA~~o protein) oa the ordinate and the percent protein

content on the abscissa . The distribution with variabilities for each of the substances measured is given in Table I. Percentages refer to amounts is each fraction compared to that in the 600 g - 10 min supernatant; i. e . 10 500 g 10 min sediment + SN . In aucroae -D 20, FI contained moat of the G 6 Pase, 7Bolo

was found in FII and the enzyme was too low to measure in FIII . Upon re-

puriFacation, G6 Pase in FII can be reduced in half . Relatively large amounts of sedimeatable NA were found in both FII (21 %) and FIII (33 %) . Although a similar distribution of NA was found in sucrose-Fî2 0 gradieata (Fig . 2), FII which contained 27 % of the NA was contaminated with 35 % of the microsomal marker G 6 Pase . It has been shown that chromaffin granules from adrenal medulla take up

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D20 preferentially when compared to certain other subcellular components (17), The present data indicate that this is also true for NA vesicles from splenic nerve trunk, which apparently become preferentially more dense is heavy water than do microsomee . NA Contents of Nerves and Purified Fractions . The NA content of desheathed splenic nerve trunk was 9. 5 I+g~g wet weight (average 6 determinations, range 5 .3-17. 0), in accordance with earlier findings (see 6) . From this amount 2~3

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could be recovered as particulate NA after removing the 600 g sediment . The distribution of NA vesicles oa the gradient indicates a wide density spectrum for the storage vesicles (Fig. 3, Table 1). The SA ie highest inF1II; i. e, average 3 . ~4 ~g NA~mg protein (range 1. 4-5. 7). On the basis of 67 ~Jo sedimeatable NA of the whole nerve (i_ e. 0. 07 ~ g NA~mg protein), it represents a 50-fold (range 20-80) purification is the average ezperimeat. It is a 4-7-fold purification over the NA vesicle preparations which have been previously reported (1-6, 18; Lishajko, personal communication) . FII had a SA of 1. 4 ~, g NA~mg protein . Thus, 55 % of the sedimentable' NA can be recovered is FII + FIII with a SA of 2, 7 w g NA~mg protein. Distrsbution of Marker Enzymes . The distribution of marker enzymes among the various fractions from D20 gradients are given is Fig. 3 and Table 1, ßGPase activity occurs is all fractions . Approtünatelp 26

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in FII and 15 % in

FIII. These can be reduced is half by repurification. The SA of this enzyme is the purified fractions increased by a factor of 3 compared bo SN (Table 1). However, at the physiological pH 7 .2, ß-GPase activity becomes negligible in both FII and FIII after a single gradient separation. Preliminary electron microscopic ezaminatioa of the fractions makes it unlikely that the remaining enzyme activity is due primarily to contaminating lysosomes . The SA of TPPase is 6 times greater thaw that of G6Pase sad ß-GPsse in the SN (Table 1). About 10% of the TPPase remains in FII sad FIII, sad the SA of this enzyme does not increase i.n. the latter compared to the SN . With low speed Ultra Tunas homogenization, about 50% of the iaitochoadrial enzyme activity, CyOz and MAO, was removed is the 10 500 g sediment. This corresponds favorably with the 60+ % removal of fumarase activity by Schtlmana (4) st the higher 15 000 g. After high speed Ultra Tura~az homogeaizatioa, much less of the mitochoadrial enzymes occurred in the 10500 g sediment, which suggested particle breakage. The latter was supported by differential distribution patterns of CyOz (heavier, inner mitochondrial membrsae) and MAO (lighter, outer mitochondrisl membrane) . Despdte the fact

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that only 15 % of the CyOx remained in FIII, the SA increased about 3-fold compared to that is the SN . Preliminary electron micrographs substantiate the presence of small mitochondria scattered is the NA fractions . It can be concluded that a portion of the mitochondria in spleaic serve truck is not only of similar density to the NA vesicles, but also acts similarly on sucroseDZO gradients in becoming preferentially heavier compared to microsomes . Evidence for mitochoadrial uptake of DZO can be found in the literature (see 17). In preliminary experiments it was shown that the purified NA vesicle fractions (FII, FIII) have the ability to take up NA, which can be stimulated in the presence of Mgt and ATP. The uptake per unit protein is greater than in the leas pure F I. It was also shows that is F III, ATP can be hydrolysed at a rate of about 0 .08 ~M Pi~min " mg protein (see Methods) . Discussion In order to study the enaymatic properties sad the uptake, storage and secretion of NA in storage vesicles in vitro, it is desirable to have preparations relatively free of enzymes belonging to other cell components, or at least to have knowledge of the activity of contaminating enzymes . By using a special technique with heavy water-sucrose gradients, we have obtained a NA vesicle fraction essentially free of microsomal marker enzyme activity . In addition, mitochondrial, lysoeomal and Golgi markers are substantially reduced relative to the increase in NA : protein ratio. The purest NA fraction (F LII) contained on average 3 .44 Fig NA~mg protein, which represents a 50-fold purification of particle bound NA after homogenization of whole nerve trunk. It is a 4-7 fold purification over those values reported in the literature (2-6). In the only earlier total gradient centrifugation study of cell organelles obtained from epleaic nerve homogenates, the highest NA : protein achieved was 0. 5 ~. g NA~mg protein (6). This lower value may in part be due to the fact that the latter NA vesicles were first sedimeafed before layering ~n sucrose gradients .

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The NA content of our purest vesicle fraction is still more thaw 2-3 orders of magnitude below that which may exist is nerve te r.i*,~ia (19), and is purified adrenal medullary granules (8). The facts that 2/3 of the NA is the eplenic nerve trunk homogenate is sedimentable (depending oa homogeaizatioa procedure) and that st least 85 % of this can be recovered at the end of purification indicate that the sedimeatable NA is in a reasonably stable state . This relatively low NA content even after 50-fold purification strongly supports the eoateatioa that the nerve tzvak vesicles have a naturally lower NA content thaw that which ie believed to occur fn nerve terminal vesicles . Reference to this possibility has been made by others (20). Preliminary elect~oamicroscopical studies by one of us sad chromogrania analyses (Karia Hells, personal communication) substantiate that a considerable percentage of ~e particles in F III moat be NA vesicles . With this improvement is vesicle purity cad quantitative data am coatami_ amts, we are now is a position to make an interesting calcuLtioa . By coaeideriag the percentage distribution of NA, protein and the average coatamiaatioa of the ~ major marker ea:ymes (ae representative of the total contamiaaats) is the various fractions, one can set up differential equations to solve for that portion of F III which ie NA vesicle protein. The value is apprazima_ lively 18 qô, the remainder being coatamiaitioa with other protein. Thus, if the NA vesicles were 100 °J6 pure, the upper limit of NA ; protein could be estimated to be 3. 4~ (the SA) z 1. 5 (if all the nerve trunk NA were vesicle bound/ 0.18 (the

°'fo

purity) = 28 . ? F+g NA/mg protein, 1f this estimate is is the cor-

rect range, it increases the probability that there is s considerable difference is NA content between nerve trsmk and terminal vesicles . The final ezperimeatsl proof is still Lckiag because of the technical difficulties to isolate terminal vesicles of reasonable parity. Any differeabe is NA content between trunk and terminal vesicles is a crucisl point is evslvatiag the current estimate of 4-7 weeks for the life span of sympathetic NA storage vesicles (21) . This estimate is based oa a cumber

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of assumpüoas including that the truck and terminal vesicles are identical and have a full NA content (21 ). Provided the other assumptions are valid, the present estimate of the upper limit for NA in true& vesicles indicates that the life span is considerably shorter . Acknowledgement This study was supported by Research Career Program Award 5-K03-HE 05892 from the Nat. Heart Inet. and research grant 5-ROl_GM15490 from the Nat. Inet. Gea. Med . Sci . 1J. S. P. H. S. to Dr . Klein, Swedish Medical Re search Council, protect no. K70-14X-2479-03A, and Knut and Alice Wallenbergs Foundation. References 1 . U. S. von EDLER sad N. -A. HILLARP, Nature ( Load. ) 177, 44 (1956) . 2 . U. S . von EDLER, Acta~hysiol . ecand . 43, 155 (1958). 3 . R. H. ROTH, L. STJÄRNE, F. BLOOM and N. J. Gù~RMAN, J. Pharmacol. ezp. Ther. 162, 207 (1968). 4. H. J. SCHUMANN, K. SCHMIDT and A. PHILIPPU, Life Sci . 5, 1809 (1966) . _ .. 5. A. BURGER, A. PHILIPPU cad H. J. SCHUMANN, Naunya_Schniedeberg'e Arch. ezp. Path. Pharmak . 262, 208 (1969) . 6. H. HöRTNAGEL, HEIDE HARTNAGEL and H. WINKLER, J. Phyeiol . Land. ) 205, 103 (1969). 7. W. W. DOUGLAS, Brit. J. Pharmacol. 34, 451 (1968). 8. A. D. SMITH, In "The interactioa of dr am in animal cells~; e . e ,

s aad subcellular com urc , o on, p.

nte

8).

9 . U . S . von EDLER and F. LISHAJKO, Acta physiol . scand. 52, 137 (1961) 10. R. J. WURTMAN aad J. AXELROD, Biochem . Pharmacol. 12, 1439 olo X", eds . S. P. 11 . R. L. POST cad A. K. SEN, In "Methods in Enz ess, . . p. (1967). Colowick and N. A. Kaplan, Ac emic 12 . A. PELLEGRINO de IRALDI and E. de ROBERTIS, Bayer Symposium II, Cologne (1969). 13 . P. BANKS, K. HELLS and D . MAYOR, Mol. P'harmacol . 5, i 210 (1969) . 14. L. T. POTTER and J. AXELROD, J . Pharmacol . ezp. Ther. 142, 291 (1963 ).

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15 . S. H. SNYDER, I. A. MICHELSON and J . MUSSACHIO, Life Sci. 3, 965 (1964) . 16 . C, de DUVE, In "Enzyme Cytol~ ", ed . D . B. Roodya, Academic Preea, N. Y., p. 22 (hô7). 17 . P. LADURON, Thesis : Bios~athés e, Localisation Intracellulaire et Transport des Catecholaminea , er, ouvaia 18 . U. S. von EDLER, Circulat . Res . 21, Suppl. III, p. 5 (1967) . 19 . K. A. NORBERG and B. HAMBERGER, Acta physiol. stand. 63, Suppl. 238 (1964) . 20. U . S . von EDLER, In "Metabolism of the Nervous S stem", ed . D. Richer, Pergamoa gene, o , p. 21 . ANNICA DAHLSTRCSM and J. HÄGGENDAHL, Acta physiol. stand . 67, 278 (1966) .