The effects of cell isolation techniques on neuronal membrane receptors

Brain Research, 93 (1975) 337-342 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

337

Short Communications

The effects of cell isolation techniques on neuronal membrane receptors

M. G U A R N I E R I , L. S. KRELL, G. M. M c K H A N N , G. W. P A S T E R N A K AND H. I. YAMAMURA

Department of Neurology (M.G., L.S.K. and G.W.McK.) and Department of Pharmacology, (G. W.P. and H.I.Y.), Johns Hopkins Medical School, Baltimore, Md. 21295 (U,S.A.) (Accepted April 29th, 1975)

Neurons, astrocytes, and oligodendroglial cell fractions have been isolated from mature brains by several methods (reviewed in refs. 13 and 16). Despite many uncertainties in the isolation procedures, studies have shown marked similarities between the properties of in vivo and isolated ceils, such as the enrichment in oligodendroglial cells of enzymes that synthesize myelin lipids 1,2. These isolated cell fractions, prepared by methods which use both endogenous or exogenous histolytic enzymes, thus introduce an important tool for neuroscience research 15. However, histolytic enzymes affect membrane proteins such as the opiate receptor 9. Cultured cells derived from CNS tumors have been used to study morphine binding 6,tv, but there is little or no information on whether cells from normal tissue can be used to study the binding of drugs or other biomolecules. In this report we have studied the effects of cell isolation procedures on the binding activity of opiate and muscarinic cholinergic receptors. Cells were isolated from the cerebral hemispheres of 15-20-day-old SpragueDawley rats (Charles River, Boston, Mass.) according to the methods described by Poduslo and Norton 13 (Fig. 1). Brains were treated with 0.1 ~ trypsin (beef pancreas, 2 × crystallized, Nutr. Biochem. Corp. Cleveland, Ohio), 0.1 ~o collagenase (CI. Histolyticum Type 1, Sigma, St. Louis, Mo.), or without enzyme. In the latter case, brains were incubated in HAP buffer or in a Ficoll buffer as described by Iqbal and Tellez-Nagel 5. The purity and identity of the cell fractions was monitored by phase microscopy as previously described 5,13,14. Specific muscarinic cholinergic receptor binding to homogenates of the enzymetreated tissue and isolated cells (20-300/tg protein) was measured with [3H]3-quinuclidinylbenzylate (QNB), a potent muscarinic antagonist, as previously described ts e0. The integrity of the opiate receptor was evaluated by the stereospeeific binding of the opiate antagonist, naloxone 9-~2. Cellular protein, determined by the method of Murphy and Kies s, was measured by subtracting the concentration of albumin in the buffer from the total protein. For

338

PREPARATION OF NEURONS AND ASTROGLIA*

MINCE BRAIN AND INCUBATE IN HAP AND ENZYME FOR 30 OR 90 MIN?

PELLE% WASH 2X WITH HAP

NYLON (2X)

---

STAINLESS STEEL

ET/

~ CRUDE GLIAL SAMPLE 0.9 M'~ SUCROSE 1.4M; IN HAP

I

*

SPIN ,....t~ SAMPLE IN HAP IXED 3 300g [.J ~'~fi~BRANE LAYER ~ 0.90"~ IO' ~-~ 1"1 GLIA I~ MIXED CELL LAYER ~ 1.35 ~,SUCROSE t:55( tN HAP / ~ NEURONS ~1 2.00) ~, DILUTE NEURONS SLOWLY 5 FOLD WITH HAP, PELLET

C~ DEBRIS

GLIA

DILUTE GLIA SLOWLY S FOLD WITH HAP, PELLET

Fig. 1 contains a schematic illustration of the preparation of neuronal cell bodies andastroglial ceUs from rat brain. HAP refers to hexose-albumin-phosphate buffer which contains 5 % fructose, 5 % glucose, 1% albumin and 10 mM phosphate buffer, pH 6.0. The asterisk (*) indicates that whereas 30 min incubations are required to dissociate neurons, 60-90 rain incubations are used to improve the yield of astrogtia. The crude glial fraction is purified on a second gradient. these measurements, the cells were pelleted from the sucrose gradient solutions and resuspended in 0.5 mt of buffer/g of brain. Approximately 3-4 mg of cell protein were obtained per g (wet wt.) of brain incubated for 30 min with trypsin or collagenase or without enzyme according to the method of Iqbal and Tellez-NageP. Similar results were obtained by a Lowry protein determinationL The yield of cells from enzyme-treated brain depends on both the concentration of the enzyme and the length of incubation. A 30 rain incubation with 0.1% trypsin or collagenase was required to obtain 10 million neuronal cells (2-4 mg cell protein) per g of rat brain. Less than 105 neuronal cells/g of brain were obtained following 10 min incubations and at least 60 min incubations were required to obtain 105 astroglial cells. The properties o f cetls from trypsinized brain have been described previously13,14: The properties of neuronal cell bodies from collagenase-treated brains will be described elsewhere. In the first set of experiments, we monitored cell isolation procedures that effected receptor binding. The receptor activity of minced brain suspended in the cell isolation buffer varied from 5-35 moles • 10 -x5 (n = 6) and 200-600 moles • 10-1~ (n = 10) for naloxone and QNB binding respectively. The cumulative effects of various histolytic incubations and the sieving procedures used to complete the dissociation of cells are shown in Table ! for a typical experiment. Collagenase treatment diminished opiate receptor binding to a greater extent than incubations with no exogenous enzyme. The amount of activity lost in brains incubated with collagenase for 30 min varied from

339 TABLE I T I l E E F F E C T S OF I I l S T O L Y T I C I N C U B A T I O N S O N R E C E P T O R B I N D I N G A C T I V I T Y IN R A T B R A I N

Ten gram portions of minced brain were incubated with 100 ml of hexose-albumin-phosphate (HAP) buffer, pH 6.0. The collagenase and trypsin experiments contained 1 mg of the respective enzymes per ml of buffer. At various times, 20 ml aliquots of the incubation mixtures were cooled, 0-5 °C and mixed with 4 ml of ice-cold buffered calf serum. The mixture was centrifuged at 140 × g for 5 rain to sediment the tissue. The supernatant solution was discarded. The tissue was washed twice by resuspension in 20 ml of HAP buffer and centrifugation. The resultant pellet was allowed to stand on ice until all the samples had been collected. The 0 rain incubation test thus remained at 0-5 °C for approximately 2 h together with tissue-bound trypsin 3. After the 90 rain samples were isolated the tissues were analysed for stereospecific [ZH]naloxone and [3H]QNB binding activity. Values are expressed as the average of 3-4 binding measurements. The individual values varied from the average by less than 10%. Enzyme treatment

Opiate receptor: naloxone bound (moles. 10 15/mg protein)

Muscarinic cholinergic receptor: Q NB bound (moles • 10 15/mg protein)

None

Trypsin

Collagenase None

Trypsin

Collagenase

18 15 14 II 12

5 4 4 2 1

14 13 12 10 9

--320 200 280

340 -170 -160

Incubation time (rain)

0 10 30 60 90

350 --300 --

35 to 55 ~o. M u c h o f this loss m a y be a t t r i b u t e d to the heat lability o f the receptor 9 a n d the presence o f e n d o g e n o u s enzymes. T r y p s i n i z e d b r a i n routinely lost b i n d i n g activity at a rate 3-5-fold faster t h a n either o f the o t h e r treatments. W h e n minced brains were mixed with trypsin, i m m e d i a t e l y mixed with ice-cold calf serum (which c o n t a i n s a trypsin inhibitor) a n d washed, m o r e t h a n 70 ~o o f the n a l o x o n e b i n d i n g was lost. This result suggests a n d chemical m e a s u r e m e n t s have confirmed 3 t h a t trypsin, which abolishes stereospecific n a l o x o n e b i n d i n g 9, is r a p i d l y a d s o r b e d to neural tissue a n d is n o t accessible to trypsin inhibitors. O p i a t e r e c e p t o r activity was m e a s u r e d also in brains which were i n c u b a t e d in a Ficoll solution as described by I q b a l and TellezN a g e P . This m e t h o d utilizes the activity o f e n d o g e n o u s histolytic enzymes for the dissociation o f n e u r o n a l a n d glial cells. Brains t h a t were i n c u b a t e d at 37 °C in Ficoll s o l u t i o n lost n a l o x o n e b i n d i n g activity at a rate similar to b r a i n s i n c u b a t e d with H A P buffer a n d collagenase. M u s c a r i n i c cholinergic b i n d i n g was r a p i d l y inactivated by collagenase while trypsin h a d less effect (Table I). Brains i n c u b a t e d with trypsin for 30 rain retained 75-95 ~ o f their Q N B b i n d i n g activity. Thus Q N B binding was less sensitive to histolytic i n c u b a t i o n s t h a n n a l o x o n e binding. Because t r y p s i n - t r e a t e d b r a i n s retained significant Q N B binding activity the d i s t r i b u t i o n o f the m u s c a r i n i c cholinergic r e c e p t o r in m e m b r a n e a n d cell fractions f r o m trypsinized b r a i n was e x a m i n e d (Table II). A p p r o x i m a t e l y 25 ~ o f the muscarinJc cholinergic r e c e p t o r activity was lost by the histolytic t r e a t m e n t (Table II). The

340 TABLE 11 RECEPTOR ACTIVITY IN CELL FRACTIONS ISOLATED FROM ENZYME-TREATED BRAINS

Naloxone binding was measured in fractions of cortical tissue incubated with 0.1 ~ collagenase solution. QNB binding was measured in fractions of tissue incubated with 0.1 ~ trypsin. Tissues were incubated for 30 rain at 37 °C, washed, sieved and fractionated as described by Poduslo and Norton 3~. Control tissues were mixed with enzyme, then immediately washed and sieved. The fractions were stored at 0-5 °C until all the fractions had been collected. Binding activity was measured as described in Table 1. Fraction

Opiate receptor: naloxone bound (moles • lO15/mg protein)

Muscarinic cholinergic receptor: QNB bound (moles • 10 1'3~ragprotein)

Per mg protein

Total

Per rng proteh;

Total

4000 1900

0.35 0.27

175.0 121.5

945 136 <5 104

0.26 0.15 0.01 0.20

37.9 14.1 0.5 13.0

Control homogenate 8.0 Enzyme-treated h0mogenate 3.8 Gradient fractions Membrane layer 5.0 Astroglial cells 1.2 Mixed cells < 0.1 Neuronal cells 1.7

recovered activity was distributed in the crude membrane band and in the cell layers. Subsequent localization was not possible because binding was not enriched in any fraction, and more than 45 ~ of the binding activity placed on the gradient could not be recovered. The naloxone binding activity applied to the density gradient sedimented into three bands corresponding to crude membrane, the astroglial celt, and the neuronal cell layer. A fourth band containing a mixture of cells and capillary processes had essentially no binding. The specific activity of the opiate receptor in the crude membrane fraction was higher than the brain suspension applied to the gradient, Table II. This result is consonant with subcellular fractionation studies localizing the receptor in synaptic membraneslL The crude membrane fraction contains synaptic membranes sheared from cells by mechanical (sieving) dissociation. However, sieving procedures, which are c o m m o n to bulk isolation methods la,1~, release pre- and postsynaptic membranes. Thus the requirements for the bulk isolation of cells, including the loss of binding associated with the sucrose density gradients, restrict the localization of receptors. This study has shown that despite the preserved microscopic ultrastructure and enzymic processes o f isolated cell fractions, the requirements for cell isolation conflicted with the integrity of opiate and muscarinic cholinergic receptors. The binding activity o f the muscarinic cholinergic (QNB) and opiate (naloxone) receptors depended on the histolytic enzyme, the incubation time and the sucrose gradient fractionation. Opiate binding was destroyed by trypsin and partially inactivated by endogenous histolytic enzymes. Q N B binding showed marked decrease with collagenase and little change with trypsin.

341 The relation between the action of histolytic enzymes in disrupting intercellular connections and in damaging receptors is unknown. The enzymes could directly attack intercellular connections, induce endogenous histolytic enzymes, or destroy a sufficient population of cells such that the remaining cells were susceptible to mechanical disaggregation. However, the specificity of trypsin for opiate receptors and collagenase for muscarinic cholinergic receptors strongly suggests that these enzymes act on sensitive linkages in the receptor itself. Although histolytic enzymes alter the surface of cells, it is possible to study a specific receptor in isolated cells by choosing an appropriately mild histolytic enzyme for that receptor. The hypertonic sucrose gradients, which are common techniques to all methods for the isolation of specific cell fractions from brain, reduced both naloxone and QNB binding activity. Cells isolated by elutriation (counter flow centrifugation), a technique which permits cell separation in isotonic solutions, may be more informative. Once these cells have been isolated it may be possible to regenerate receptors in culture, as has been done for the nicotinic acetylcholine receptor 4. This research was supported by funds from USPHS Grant NS-10920, NS-10465, and Johns Hopkins Drug Abuse Research Center Grant DA-00266. G. W. Pasternak was supported by the Mutual of Omaha and the United Benefit Life Insurance Companies through the Insurance Medical Scientist Fund. H. I. Yamamura was supported by a Special Research Fellowship A w a r d MH-54777 from the National Institute of Mental Health.

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galactosyl diglyceride in glial and neuronal enriched fractions of actively myelinating rat brain, J. Neurochem., 22 (1974) 479-485. 3 GUARNIERI, M., GINNS, E., AND COHEN, S. R., Cells isolated from trypsin treated brains contain trypsin, J. Neurochem., in press. 4 HARTZELL, H. C., AND FAMBROUGH, D. H., Acetylcholine receptor production and incorporation into membranes of developing muscle fibers, Develop. Biol., 30 (1973) 153-165. 5 IQBAL, K., AND TELLEZ-NAGEL, I., Isolation of neurons and glial cells from normal and pathological human brains, Brain Research, 45 (1972) 296-301. 6 KLEE, W. A., AND NIRENI~ERG, M., A neuroblastoma × glioma hybrid cell line with morphine receptors, Proc. nat. Acad. Sci. (Wash.), 71 (1974) 3474-3477.

7 LOWRY,O. H., ROSEBROUGH,N. F., FARR,A. L., AND RANDALL, R. J., Protein measurements with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 8 MURPHY, J. B., AND KIES, M. W., Note on the spectrophotometric determination of proteins in dilute solutions, Biochim. biophys. Acta (Amst.), 45 (1960) 382-384. 9 PASTERNAK, G. W., AND SNYDER, S. H., Opiate receptor binding: effects of enzymic treatments, Molec. Pharmacol., 10 (1974) 183-193. 10 PERT, C. B., AND SNYDER, S. H., Opiate receptor: its demonstration in nervous tissue, Science, 179

(1973) 1011-1014. 11 PERT,C. B., ANDSNYDER,S. H., Properties of opiate receptor binding in rat brain, Proc. nat. Acad. Sci. (Wash.), 70 (1973) 2243-2247. 12 PERT, D. B., SNOWMAN,A. M., AND SNYDER,S. H., Localization of opiate receptor binding in synaptic membranes of rat brain, Brain Research, 70 (1974) 184-188.