Subcellular fractionation of bovine ganglion stellatum: co-storage of noradrenaline, Met-enkephalin and neuropeptide Y in large ‘dense-cored’ vesicles

Brain Research, 442 (1988) 124-130

124

Elsevier BRE 13312

Subcellular.fractionation of bovine ganglion stellatum: co-storage of noradrenaline, Met-enkephalin and neuropeptide Y in large 'dense-cored' vesicles Erik Bastiaensen, Bernard Miserez and Werner De Potter Laboratory of Neuropharmacology, Departmentof Medicine, Universityof Antwerp (UIA), Wilrijk (Belgium) (Accepted 18 August 1987)

Key words: Sympathetic ganglion; Co-existence; Enkephalin; Neuropeptide Y; SubceUular fractionation; Vesicle

The subceUular localization of noradrenaline, Met-enkephalin and neuropeptide Y was studied in homogenates of bovine ganglion stellatum. After differential centrifugation most of the noradrenaline (70%) was found soluble, while both neuropeptide Y and Metenkephalin were sedimented for more than 65%. However, the 3 substances co-sedimented mainly in the microsomal fraction. The microsomal fraction was further analyzed by differential and equilibrium density gradient centrifugation. In both types of gradient, Met-enkephalin and neuropeptide Y were found in the more dense region of the gradient, coinciding with the main peak of noradrenaline. In this fraction, the molar ratio of Met-enkephalin to noradrenaline was 1:95. The corresponding molar ratio for neuropeptide Y to noradrenaline was 1:253. These results indicate that neuropeptide Y and Met-enkephalin are stored with noradrenaline in 'heavy' or large 'dense cored' vesicles in the cell bodies of sympathetic neurons of bovine ganglion stellatum. We present here for the first time biochemical evidence for the co-localization of neuropeptides and a classical transmitter in a neuronal cell body.

INTRODUCTION

Several examples of co-existence of peptides and amines, both in the peripheral and central nervous system, have been observed in a relatively short time 15, The possible occurrence of two or more transmitters in the same neuron raises the question of their subcellular storage sites. The subcellular localization of classical transmitters is considered to be within the storage vesicles. The intracellular storage site for most neuropeptides has not yet been extensively characterized. For noradrenaline (NA), there are two types of vesicles; large (diameter 70-100 nm) and small (50 nm) 'dense cored' vesicles. Electron-microscopic immunocytochemical studies have shown Met-enkephalin (ME)-like immunoreactivity in large 'densecored' vesicles in brain, adrenal medulla, vas deferens and sympathetic ganglia 3.7. By the same technique, neuropeptide Y (NPY) was shown to occur in

large 'dense-cored' vesicles in braint6. Evidence from centrifugation studies of bovine splenic nerve 9.t3,~s, bovine 3'14 and rat 9 vasa deferentia, indicates storage of enkephalins and NPY in large 'dense cored' vesicles. There have been only a few detailed studies on the subcellular distribution of NA in sympathetic ganglia2'5'6. So the question of how peptides and even NA itself are stored in sympathetic ganglia is largely unresolved. In this article, experiments are described in which the distribution of NA between fractions obtained by differential and density gradient centrifugation of homogenates of bovine ganglion steUatum is compared with the distribution of NPY and ME. MATERIALS AND METHODS

Subcellularfractionation Bovine ganglia stellata were obtained from the

Correspondence: W. De Potter, Laboratory of Neuropharmacology, Department of Medicine, University of Antwerp (UIA), Universiteitsplein 1, B2610 Wilrijk, Belgium, 0006-8993/88/$03.50 (~ 1988 Elsevier Science Publishers B.V. (Biomedical Division)

125 slaughterhouse within 30 min postmortem and were immediately put in an ice-cold 0.25 M sucrose/5 mM Tris-HC! (pH 7.3) buffer. The tissues were dissected free from contaminating tissue, cut into small pieces with scissors, and then homogenized in an Ultra-Turrax (Janke and Kunkel, F.R.G.) at half-maximal speed for 60 s in 5 vols. of 0.25 M sucrose buffer. The homogenate was centrifuged at 500 gmax for 10 min, the pellet was resuspended in sucrose buffer, homogenized and centrifuged as before. The supernatants were pooled and filtered over surgical gauze. The filtrate was centrifuged for 10 min at 2000 gmax, giving a pellet Pl and again at 10,000 gmax for 20 min, giving a pellet P_,. The resulting supernatant was further centrifuged at 140,000 gmax for 45 min which gave a supernatant SN 3 and a pellet P3 (microsomal fraction). This pellet was used for further analysis by two techniques of density gradient centrifugation. For isopycnic gradient centrifugation, the pellet was resuspended in 0.25 M sucrose and layered onto a 10.5 ml sucrose density gradient. The density gradient consisted of a bottom step of 0.5 ml 2 M sucrose and above this a linear gradient from 0.3 to 1.6 M sucrose. The gradient was centrifuged at 272,0011 gmax for 110 min in a Beckmann ultracentrifuge with a SW41 swing out rotor. For the differential gradient centrifugation 0.5 mi of the resuspended microsomal fraction was layered on top of a linear sucrose gradient ranging from 0.3 to 0.8 M buffered sucrose on a 2 M sucrose cushion. The tubes containing these gradients were centrifuged at 272,000 g.,,,x for 3{I rain. After centrifugation 12 fractions were collected starting from the top with a Buchler autodensi-flow llc and aliquots were taken for analysis of NA, ME, NPY and protein. The density of the fractions was determined using a refractometer.

NA The NA content of the fractions was determined electrochemically after separation by high-performance liquid chromatography !1. Dihydroxylbenzamine was used as an internal standard.

Protein Proteins were determined according to the method described by Bradford i with bovine serum albumin as standard.

Chromatography A fast protein liquid chromatography (FPLC) system (Pharmacia) was used which was equipped with a reversed-phase column (PEP-RPC HR 5/5). All eluentia were degassed and filtered prior to use. Samples were centrifuged or filtered. For identification of ME- or NPY-immunoreactivity, fraction 8 of the isopycnic gradient was taken and prepurified over Sep-Pak C-18 cartridges (Waters Inc., MA, U.S.A.). The vacuum-dried methanol elute was resuspended in 0.1% trifluoroacetic acid (TFA), centrifuged and injected on a PEP-RPC HR 5/5 column. For analysis of ME, elution was with a linear gradient of 10-40% acetonitrile in 0.1% TFA over 90 min at a flow rate of 0.5 ml/min. For NPY the elution was with a gradient of 20-50% acetonitrile in 0.1% TFA over 60 min at a flow rate of 0.5 ml/min. Standard peptides were chromatographed under identical conditions. Between consecutive runs of standards and tissue extracts the column was washed with a 0 to 100 to 0% gradient in 2-propanol containing 0.1% TFA to avoid contamination. One-minute fractions were collected and dried under reduced pressure prior to radioimmunoassay (RIA).

ME and N P Y For both peptides, aliquots from differential and density gradient centrifugation were concentrated and purified prior to RIA over Sep-Pak C-18 cartridges. The ME antiserum (UCB Bioproducts, Belgium) was used in a 1/16,000 dilution. It exhibits ~, 7% cross reactivity with Leu-enkephalin and less than 0.1% with other small peptides. The sensitivity of the assay was 5 fmol/2001d with an IC50 (50% of maximal binding) of 40 fmol/200 ld. For this RIA a saline buffer (125 mM sodium phosphate, pH 7.4, 4{I mM NaCI, 0.05% sodium azide) containing {I.125% bovine serum albumin was used. The incubation was carried out at 4 °C for 18-24 h in a total volume of 200 !d, which comprised 1{111F~Iof the sample or standard, 50 Itl of the antibody and 511:~1 of the tracer. Bound and free iodinated ME (Amersham Int. U.K.) were separated, after a 311min incubation at 4 °C with 1 ml of a 20% polyethylene glycol solution, by a 2{I min centrifugation at 3500 g. For the NPY RIA a 51/mM sodium phosphate (pH

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Fig. l, Reversed-phase FPLC of ME-like (A) and NPY-like (B) immunoreactivity extracted from the fraction 8 of an isopycnic gradient from bovine ganglion stellatum. Conditions are described in Materials and Methods. LE, Leu-enkephalin; O, octapeptide MEArg6-Gly7-LeuS;H, heptapeptide ME-Arg6-Phe7. NPY is the elution position of synthetic porcine NPY.

127 7.4) buffer containing 0.1% Triton X-100 and 50 mM NaC! was used. The NPY antiserum (Amersham Int. U.K.) recognizes the C-terminal region and it has a cross reactivity of 30% with peptide YY and less than 0.1% with pancreatic polypeptide. The assay has an IC50 value of 110 fmol and a sensitivity of 5 fmol in a total volume of 300/d. Incubation was carried out for 2 or 3 days, and precipitation was as described for the ME RIA. RESULTS

Identification of ME and NPY with reversed-phase FPLC Each fraction collected after reversed-phase FPLC of an extraction sample of fraction 8 of the isopycnic gradient was assayed for ME content by radioimmunoassay. As can be seen in Fig. IA most ME-like immunoreactivity detected was localized in fractions corresponding with the retention time of authentic ME. The small peaks in other fractions are due to the cross reactivity of the antiserum with other enkephalins. In a similar experiment (Fig. 1B) NPY-like immunoreactivity occurring in bovine ganglia stellata extracts was shown to have the same retention time as synthetic porcine NPY.

Homogenization and differential centrifugation After differential centrifugation 3 particular fractions and a final supernatant were obtained. Table i gives the absolute value of the determinations of NA, protein and peptides in the total homogenate as well as the results in terms of the proportion of the total amounts of these constituents in each fraction. The molar ratio of ME to NA and NPY to NA was, re-

spectively, 1:566 and 1:999 in the total homogenate and 1:213 and 1:421 in the microsomal fraction (P3)When expressed as % sedimentable material over the total amount recovered in PI --I- P2 -t- P3 + SN3, the corresponding values were 32.3% for NA, 14.5% for protein, 66% for NPY and 72.0% for ME. The relative purification, as expressed as the ratio of the amount per mg protein in P3 t o that in the total homogenate, is 2.4 for NA, 6.3 for ME and 5.6 for NPY. Although it was noticed that a relatively large percentage (30-35%) of the sedimentable NA is also present in the P2 fraction, in analogy with our earlier experiments on splenic nerves and dog spleen 4 only the P3 fraction was used for gradient centrifugation experiments. The degree to which labeled tracer peptides added to the total homogenate remained intact, was monitored. After 6 h of incubation at 0 °C the samples were analyzed by reversed-phase FPLC. For ~-'51NPY 54.0 + 7.2% (n = 3) of the added amount still eluted in the original position. For ME this value was much lower, i.e. 18.1 + 3.5% (n = 3).

Density gradient centrifugation Two types of gradient centrifugation techniques were used i.e. differential (0.3-0.8 M sucrose, gradient A) and isopycnic gradient centrifugation (0.3-1.6 M sucrose, gradient B) by which the subceilular particles are separated on the basis of their sedimentation velocity and equilibrium density, respectively. The subcellular distribution of NA, peptides and protein in density gradients from bovine ganglia stellata expressed as the percentage of totally recovered material in the gradient is shown in Figs. 2 (gradient A) and 3 (gradient B). The result of differ-

TABLE I

Differential centriJhgation of bovine ganglion steilatum. Distribution of NA, ME, NPY and protein in fractions obtained by differential centrifugation of homogenates The proportion of each constituent in a flaction is given as a percentage of the total recovered amount (Pt + P-" + P3 + SN3)" The actual recoveries given in the last line are expressed as a percentage of the total homogenate (TH). Results are mean + S.E.M. of 5 experiments.

TH Proteins NA ME NPY

20.1 8.3 14.7 8.3

Pt + + + +

1.8 mg 1.3 nmol 2.7 pmol 1.6 pmol

2.3 3.1 3.6 5.9

P2 + + + +

0.2 0.4 0.2 0.8

4.7 + 11.3 + 21.4 + 17.8 +

P3 1.4 1.1 1.2 1.7

7.4 17.7 46.8 41.7

S., + + + +

1.7 1.4 2.1 1.6

85.5 67.7 28.0 34.1

Recovery + + + +

!.6 1.2 1.4 1.8

94.1 88.1 89.7 87.1

+ + + +

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Fig. 2. Subcellular distributions of NA, ME, NFY and protein in differential density gradient of a microsomai fraction, obtained by differential centrifugation of a ganglion stellatum homogenate. On the ordinate, totally recovered substance is given as the percentage of the totally recovered material in the gradient. On the abscissa, density gradient fractions l-12 are given, corresponding to the following molarities: l (0.28 M), 2 (0.34 M), 3 (0.37 M), 4 (0.4 M), 5 (0.43 M), 6 (0.51 M), 7 (0.57 M), ~ (0.62 M), 9 (0.65 M), l0 (0.70 M), l 1 (0.78 M), 12 (0.81 M). Recoveries of NA 77%, NPY 81%, ME 86% and protein 85%. Results are expressed as the mean of 4 experiments +_ S.E.M.

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Fig. 3. Subcellular distributions of NA, ME, NPY and protein in equilibrium density gradient of a microsomal fraction obtained by differential centrifugation of a ganglion stellatum homogenate. On the ordinate, totally recovered substance is given as the percentage of the totally recovered material in the gradient. On the abscissa, density gradient fractions 1-12 are given, corresponding to the following molarities: 1 (0.27 M), 2 (0.39 M), 3 (0.55 M), 4 (0.68 M), 5 (0.80 M), 6 (0.90 M), 7 (1.05 M), 8 (1.14 M), 9 (1.21 M), 10 (1.34 M), 1 ! ( 1.42 M), 12 ( 1.56 M). Recoveries of NA 78%, NPY 83%, ME 87% and protein 84%. Results are expressed as the mean of 6 experiments _+ S.E.M.

129 ential gradient centrifugation (Fig. 2) shows that NA, ME and NPY are present in particles with the same sedimentation rates. The result of the isopycnic gradient centrifugation (Fig. 3) further shows that these particles also equilibrate at the same density i.e. 1.15 M sucrose. From previous studies, this fraction is known to contain large dense-cored vesicles. In isopycnic gradient no clear peak of NA in the less dense region of the gradient was found. The considerable amount of NA accumulating in the upper regions of the differential gradient was not particle bound, and was presumably derived from vesicles damaged by resuspension or by leakage. When the 6 upper fractions of this gradient were recentrifuged to spin down intact NA vesicles, most of the NA was found to be soluble (results not shown). The specific activity of NA, NPY and ME in the different fractions of isopycnic gradient can be seen in Table II. The enrichment of these compounds relative to the total homogenate is also shown. NA is enriched 3-fold in fraction 8, NPY is enriched 12.5-fold, whereas ME is enriched 17.7-fold. The molar ratio of NPY to NA in the peak fraction is 1:253. The corresponding molar ratio for ME to NA is 1:95. DISCUSSION It can be seen from Table l that upon differential TABLE II Relative enrichment in fractions of d(fferential centrifugation and isopycnic centrifugation gradients, expressed as the ratio of the specific activity (pmoi/mg of protein) in fraction to that of homogenate Fraction

NA

ME

NPY

TH PI P,

1 1.4 2.4 2.4 2.0 1.7 2.6 !.0 1.2 2.2 2.9 3.0 2.8 2.1 2.4 2.7

1 1.6 4.6 6.3 0.7 0.1 0.4 0.2 1.2 2.5 12.0 17.7 11.0 7.0 3.4 6.5

1 2.5 3.7 5.6 0.2 0.2 0.1 0.1 0.5 3.9 7.8 12.6 9.5 6.0 3.8 9.1

1 2 3 4 5 6 7 8 9 10 11 12

centrifugation and in spite of a carefully optimized homogenization procedure, almost 70% of the NA was not sedimentable. This result agrees well with earlier studies on bovine and cat sympathetic ganglia, where most of the noradrenaline was found soluble 2"5"6. According to our previous studies-" there is probably very little difference between ganglia and the nerve axons in terms of the amount of particle breakage caused by homogenization. For this reason, the difference in NA distribution observed in the ganglia and axons is most likely due to real differences in the state of the NA-storing particle itself. Indeed, it seems possible, as proposed by H6kfelt ~-'. that the so-called "free" or non-granular-bound NA ~: found in the supernatant of subceilular fractions of sympathetic ganglia, might be derived to a large extent from labile granular-bound NA such as that localized in the tubular reticulum, a third NA storage cell organelle. In contrast with NA, NPY and ME were both sedimentable for 66 and 72%, respectively. Since we noticed in control experiments a considerable proteolysis of added ME the percentage of the peptide found in the final supernatant may be somewhat underestimated. More importantly, however, is the fact that for each of the 3 substances 55-60% of the total amount sedimented is found in the microsomal fraction, indicating that the particles containing these substances have a similar size. For isopycnic gradients the present analysis demonstrates a main peak of NA in the high-density region (1.15 M sucrose). In fact, the NA distribution found in bovine ganglion stellatum is similar to that found in splenic nerve axon, but different from the NA distribution found in organs such as spleen and vas deferens. These tissues, containing terminal parts of sympathetic neurons, also show an NA peak in the less dense region of the gradient 3"~. corresponding to the small dense-cored vesicles. The possibility that similar but more labile vesicles corresponding to the small dense-cored vesicles are also present in bovine ganglion stellatum -''~7cannot be excluded. In addition and more importantly, we have now shown that in isopycnic gradients of ganglion stellaturn ME and NPY have a similar distribution, with a peak in the same high-density fraction as NA. These results, taken together with those of the differential

130 and differential gradient centrifugation, indicate that these 3 substances are stored in organelles of the same buoyant density and the same sedimentation velocity, and that NPY and M E may coexist with N A in the large dense-cored vesicles known to occur in these fractions 4. In a recent immunohistochemical study of Fried et al. l0 it was shown that the majority ( > 9 0 % ) of the cell bodies of bovine coeliac ganglion neurons contain both NPY, M E and NA. Only a few NA-positive but NPY- or ME-negative cell bodies could be detected. The most logical explanation therefore would be that NPY and M E are both present in the same vesicles which contain N A and which are found in the majority of the cell bodies, i.e. the large dense-cored vesicles. Admittedly it is sti!J possible that within a given cell body there are vesicles which contain either N A , NPY or M E separately but which all have similar sizes and equilibrium densities.

The only way to disprove the latter hypothesis with certainty would be to carry out immunohistochemical studies at the electronmicroscopic level. Our results concerning the distribution of ME are consistent with similar subcellular studies on bovine splenic nerve l°'13"ls and bovine vas deferens 3"t4. The distribution of NPY is similar to recent published studies on rat vas deferens 9, cat spleen 8 and bovine splenic nerve to. Thus, it seems that co-storage of M E and/or NPY with N A in large 'dense-cored' vesicles is a general feature of sympathetic neurons containing these peptides. ACKNOWLEDGEMENTS This work was supported by a grant of the Queen Elisabeth Medical Foundation 1987. E.B. and B.M. were supported by a grant from I . W . O . N . L .

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