Localization of noradrenaline and dopamine-β-hydroxylase in C1300 mouse neuroblastoma a biochemical and electronmicroscopic study

Localization of noradrenaline and dopamine-β-hydroxylase in C1300 mouse neuroblastoma a biochemical and electronmicroscopic study

Life Sciences, Vol, 23, pp . 2665-2674 Printed in the U .S .A . Pergamon Press LOCALIZATION OF NORADRENALINE AND DOPAMINE-ß-HYDROXYLASE IN 01300 MOU...

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Life Sciences, Vol, 23, pp . 2665-2674 Printed in the U .S .A .

Pergamon Press

LOCALIZATION OF NORADRENALINE AND DOPAMINE-ß-HYDROXYLASE IN 01300 MOUSE NEUROBLASTOMA A BIOCHEMICAL AND ELECTRONMICROSCOPIC STUDY(1) W . P. De Potter, N. H. Fraeyman, J. W . Palm(Z) and A, F. De Schaepdryver Heymane Institute of Pharmacology, University of Ghent Medical School, De Pintelaan 135, B-9000 Cheat, Belgium (Received in final form November 6, 1978) Summary Investigations were carried out in order to obtain biochemical and electronmicroscopic information on the storage of noradrenaline (NA) and dopamine-ß-hydroxylase (DßH) in 0 mouse neuro1300 bla atoms . By application of two different gradient centrifugation methods only one population of NA vesicles, i. e. heavy NA containing vesicles could be demonstrated. Electronmicroscopic investigation of the tumor revealed the presence of large dense cored vesicles . Electronmicroscopic investigation of gradient fractions further showed that large dense cored vesicles were only present in those fractions of the gradient containing heavy vesicles . The distribution of DßH in relation to the distribution of NA after gradient centrifugation differs from what has been observed in peripheral noradrenergic neurone, In C mouse neuroblastoma the larger part of the enzyme is associaté~with the plasma membrane . Electronmicroecopic studies of human neuroblastoma have shown that one of the characteristic ultraetructural features of these tumors is the presence of large dense cored vesicles which are thought to contain catecholamines (1 ). This view is rather well supported by the results of differential centrifugation experiments which showed that NA ie associated with subcellular structures (2, 3, 4), thus suggesting the presence of NA containing vesicles . More direct evidence for their presence has been obtained by two groups of workers (3, 4) who, in using density gradient centrifugation, were able to characterize NA vesicles . These vesicles had a similar density as the heavy vesicles found in bovine sympathetic ganglia (5, 6) sad eplenic nerves (7, 8, 9, 10). (1) Aided by a research grant from the A. S, i~ . K. -Cancer Fund (Belgium) . (2) Heymaas Foundation Research Fellow, on leave from the Department of Pharmacology, College of Medicine, Ohio State University, Columbus, Ohio, U. S . A, 0300-9653/78/1231-2665$02,00/0 Copyright (c) 1978 Pergamon Press

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A thorough biochemical study of the NA storage vesicles in neuroblastoma as done for adrenergic nerves (10) - ie made difficult by the rarity of this tumor. It was therefore felt that the CL~00 mouse neuroblastoma, a round cell transplantable tumor of sympa ehc cells (11, 12, 13) which grows relatively rapidly and which is also able to synthesize and store NA (14), might be a good substitute . Electronmicroscopic studies of C 13p~ mouse neuroblastoma have already revealed the presence of dense cored"vesicles (15, 16, 17), Although the composition of these vesicles is so far completely unknown (1 7), there ie biochemical evidence that a large part of the NA in the tumor ie associated with eubcellula.r structures (12) . The present study was performed in order to characterize the NA storing organelles and to investigate whether these NA containing structures correspond to the morphologically demonstrated large dense cored vesicles . Materiale and Methode 1, Biochemical. The tissue used in this study was the solid mouse 01300 neuroblaetoma . The tumor was obtained from Jackson Laboratories (Bar Harbor, Maine, U. S. A, ) and was further maintained in our laboratory by subcutaneous transfer in 4 to 10 weeks old A~J mice of both sexes. About two weeks after the inoculation, the mice were killed by cervical dislocation and the tumor dissected free from the surrounding tissue . The tissue was rinsed three times in ice-cold 0.25 M sucrose, chopped finely with a knife, suspended in 4 volumes of ice-cold 0. 25 M sucrose buffered with 5 mM trisHCl pH 7. 3 and homogenized in a motor driven Potter-Elvehjem homogenizer (Kontes, Vinela.ad, New Jersey, U. S. A, ) using a Teflon pestle (clearance 0. 12 mm). The homogenization was continued until the pestle had been passed up and down once . The homogenate was submitted to differential centrifugation in two subsequent steps : 3, 000 g for 10 min (Sorvall) followed by S8, 000 g for 45 min (Spinco L50 ) . This procedure yields three fractions, indicated by sediment 1 (mainly composed of unbroken material, nuclei and the larger mitochondria), sediment 2 (or microsomal fraction) and a supernatant. Sediment 2 was subfractionated using two types of gradient centrifugation . Both gradients were spun in the SW 40 rotor of the Beckman ultracentrifuge . Differen_tial~radient centrif_u~ation. Sediment 2 was reeuspended in the 0.25 M éucrose solution ~0. 5 ml%g original tissue) and applied oa top of a linear 0 . 3 to 0, 8 M sucrose gradient . The bottom of the tube contained 1 . 0 ml of 2, 0 M sucrose. The gradient contained 5 mM trie -HCl pH 7. 3 and was centrifuged for 40 min at 198, 000 g av' Ec~uilibr_ium deneit~!radient centrifu~atio_n. Sediment 2 was re suspended in 0, 4~ M sûcrôéè ~0 . 5 ml]g ôriginâf tiseue~ and 0, 75 ml of the suspension was layered on top of a 0, 5 to 1 . 7 M sucrose gradient which was constructed on top of a 0. 5 ml cushion of 2 M sucrose (all solutions contained 5 mM trie -HCl pH 7 . 3), The total volume (sample + gradient + cushion) was 12 . 5 ml, The gradient was spun for 150 min at 198, 000 g av' After centrifugation, 12 fractions of 1 ml each were obtained by pumping a 2 M sucrose solution through the bottom of the tube and collecting by overflow (ISCO - model 640 density gradient fractiona.tor), Aliquots of these fractions were used for the determination of noradrenaline (1 S), ß-glucuronidase (1 q), monoamine oxidaee (MAO) (20), glucose-6-phosphatase (21), inosine diphosphataee (22), alkaline phoephatase (23), '5-nucleotidase (24) and proteins (25) . DßH was routinely determined ae follows

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The incubation mixture contained 1, 5 mM tyramine, 6 mM ascorbic acid, 6 mM fumaric acid, 0, 8 mM tranylcypromine sulfate, 0, 1 mM p-chloromercuribenzoate (PCMB), 3, 200 unite of catalane, 0. 2 °f° (W~V) Triton X-100, 50 mM tria-HCl buffer (pH b) and a 200 Wl sample in a total volume of 0 . 4 ml . The tyramine, aecorbate and fumarate solutions were adjusted to pH 6 with NaOH . After 40 min of incubation at 37° C the reaction was stopped by addition of 100 ~~1 of 12 . 5 °f° (W~V) trichloroacetic acid and after centrifugation at 2, 000-3, 000 rev. min for 10 min, the supernatant extracted with 2 ml of ethylether (saturated with water) . The extraction (to remove trichloroacetic acid) was repeated four times, The remaining ethylether wan then removed by bubbling a stream of nitrogen through the mixture, The second step of the method was initiated by adding 200 ~.l of thin treated sample to a mixture containing 100 N1 of purified PNMT, 2, 7 ~IVI S-adenosyl-L- [ methyl 14 0 1 methionine (0, 1 ~.Ci) and 0. 5 M trio-HCl buffer (pH 8. 6) . The final volume was of 400 N1 . The mixture wan incubated at 3 7° C for 30 min and the reaction stopped by addition of 1 ml f 0, 5 M sodium-borate buffer (pH 10) and 3 g of sodiumchloride . The [~ 4C ] -labeled N-methyl octopamiae (synephrine) was extracted with 6 ml of a toluene-ieoamylalcohol mixture (3~2, V~V), After centrifugation 4 ml of the organic phase wan transferred to a vial for radio activity determination. In addition to a blank, standard values were obtained with each assay by adding 40 ng and 80 ng of octopamine to a complete assay mixture and these samples were carried through the procedure, In some cases the method of Molinoff et al, (26), in which CuS04 ie used to inactivate endogenous inhibitors, has been used, The distribution of NA and enzyme activities ie obtained by expressing the value of each fraction as a percentage of the sum of all fractions of the gradie¢, 2, Electronmicroscopy. Tumors were excised at different times after inoculation and small pieces immediately immersed into ice-cold 6 °Jo glutaraldehyde in sodium phosphate buffer (pH 7. 2 0. 1 M) for 1 hr . Postfixation was carried out with 1 °f° osmium tetroxide, The fixed tissue was dehydrated in an alcohol series and propylene oxide and embedded in a Spurr Low Viscosity Embedding Medium (SEM). Semithin and ultrathin sections were cut with a LKB ultrotome III. Samples from gradient centrifugations were diluted with ice-cold 6 °f° phosphate buffered glutaraldehyde and centrifuged (88, 000 for 60 min) . Further gav treatment of the resulting pellet was as for the tiesues . The sections were viewed in a J. E, M. 100 B (JEOL) electronmicroacope . Re culte Differential centrifugation . Homogenates of 0 mouse neuroblastoma 1300 contain NA and several enzymes which have been shown to be characteristic for different aubcellular particles or structures (Table I), Thie table gives the NA and enzyme contents of the tumor as well as the distribution of these constituents amongst the aubcellular fractions obtained by differential centrifugation . It can be seen that a large part of NA sediments in fractions 1 and 2 (81, 6 _+ 5 . 7 °fe), which is in agreement with previous observations on human neuroblastoma (4). The sedimeatable form of NA moat probably represents the NA storage vesicles whose distribution appears to be different from that of marker enzymes ouch as ~ -glucuronidase (lysosomea), MAO (mitochondria), glucose-b-phoephatase (endoplaemic reticulum) and alkaline phosphatase (plasma membranes),

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TABLE I Distribution of Constituents of Cf3 Mouse Neuroblastoma Among Fractions 00 Obtained Upon Differential Centrifugatidn of Tumor Homogenates . Enzyme

No . of Experimente

NA

7

ß -Glucuronidase

3

MAO

6

Glucose-6Pass

3

Alkaline phosphatase

6

DßH

3

Proteine

7

Absolute Value in Total Homogenate

Distribution (%) Fraction 2

1

+

1, 4 0,2

+

17. 1 2,8

+

+

0, 04 0, O1

+

60, 8 6, 5

+

+

0. 54 0. 07

+

67. 7 6. 0

27, 6 + 2. 4

+

45 . 2 5. 6

+

+

5. 1 0, 4

+

1 92, 7 4S . 4

+

721, 5 70, 7

+

114 . 0 5. 8

46 . 7 + 3. 9 + +

30, 7 6, 3 51, 6 0, 9

Recovery ( °fo)

64 . 5 5 .0 7, 1 0, 9

3 18 . 9 + 1 .5

+

79 . 0 4.6

+

32, 1 7, 2

+

80, 3 7, 4

+

4, 5 1. 9

+

90 . 6 2, 2

34 . 4 1, 6

20 . 0 + 4. 3

105, 6 + 22 . 4

2 7. 9 + 2, q

25 . 4 ± 1, 8

+

90, 9 4. 4

38 . 5 + 4. 1

+

103 . 2 12, 0

39 . 6 0, 8

+

95 . 1 1.8

30, 8 + 2. 3 +

B. 8 0. 9

+

Units : NA : F~g~g wet weight of tissue Enzymes : units~g wet weight of tissue Proteins : mg~g wet weight of tissue Results are the mean value _+ S. E, M. DßH activity is determined using the above described PCMB method . DßH is also present in a sedimentable form, at least 61, 5 _+ 8. 6 °fo under the present centrifugation conditions ; and it is noteworthy that, comparatively to NA, a larger percentage of DßH is present in fraction 1, Gradient centrifugation . Gradient centrifugatione were carried out in order to further characterize these NA storage vesicles . Upon equilibrium density gradient centrifugation (Fig . 1) NA appears to be associated with particlee which are slightly less dense than lyeosomes (marked by ß-glucuroaidase), about as dense ae mitochondria (marked by MAO) and more dense than endoplasmic reticulum (marked by glucose-6-phoephataee and inosine diphosphatase) and plasma membranes (marked by alkaline phoephatase and ' S-nucleotidaee), Figure 1 further shows that the largest part of DßH is found in the low density region of the gradient where it almost completely parallels the distribution of alkaline phosphatase and '5-nucleotidase . However, it ie well known that light NA vesicles - which are mainly found in adrenergically innervated organs - also equilibrate in this region of the gradient . There ie no indication for the existence of such light NA vesicles in this tumor but the possibility still exists that DßH may be associated with light - eventually (relatively) empty - NA vesicles,

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î0~

o~

Noradrenaline Storage

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,~

2

~

`~--,

r~~

ie

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o

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iW

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FIG.

1

Equilibrium density gradient centrifugation of a NA-enriched pellet of 01300 mouse neuroblaetoma. To check this eventuality differential gradient centrifugation experiments were carried out. Such a centrifugation procedure, ae illustrated in studies on rabbit iris and dog spleen (Fig, 2), allows a clear separation of heavy and light NA vesicles since the first ones are found on the bottom whereas the second ones accumulate in the upper fractions of the gradient . From Figure 2 it can be seen that no NA and only low amounts of DßH are found in the upper parts of the gradient. In order to check whether the distribution of DßH was real, i. e. not dependent upon the method used, DßH activity was also determined by the method of Molinoff et al . (26) . It is noteworthy that the distribution of DßH was e~ctly the same for the two methods used . Additionally, prior dialysis also did not affect the distribution of DßH or of any of the other marker enzymes . The recoveries of the different enzymes determined after both equilibrium and differential gradient centrifugation varied between 85 and 110 9e .

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60 , Sa

10-~

0

' J

-,

~Î -ZL-~~-r-r-~^ ~crLr -' 0

1

i 10 12 FRACTION NUMBER DIRECTION OF SEDIMENTATION

FIG.

2

Differential gradient centrifugation of a NA-enriched pellet of C mouse neuroblaetoma, rabbit iris and dog spleen . In mouse ne iôû4astoma C1 30p only heavy vesicles are found, in contrast to rabbit iris and dog spleen, in which both light and heavy vesicles are present, Electronmicroscopv . Although the electronmicroscopic investigations were mainly done with the aim of finding a correlation between dense cored vesicles and NA storage particles isolated by cell fractionation techniques, it should be noted that the fine structure of the cells of 0130a neuroblastoma (Fig . 3) resembles, in many respects, that of isolated round cells grown in tissue culture (17), Some large dense cored vesicles with a mean diameter of _+ 1500 a~. were present; but no small dense cored vesicles could be detected ; C shaped virallike particles were very frequently found (Fig . 3A) . Simila.r results have been reported for isolated neuroblastoma cells (1~), After gradient centrifugations (equilibrium as well as differential gradient centrifugation), several fractions were collected to obtain sediments for electronmicroecopic investigation. Fig, 3B shows the denser region of the equilibrium density gradient (fractions 7, 8 and 9), whereas Fig. 3C represents fraction 12 of the differential gradient : in both cases large dense cored vesicles can be seen . In other fractions of the gradient, including fractions 3, 4, 5 of the equilibrium density

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FIG.

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3

Electronmicroecopic picture of (A) C tumor (x 26, 730) ; (B) tubes 7 and 8 from the equilibrium density gradient ~x~5, 640) and (C) tube 12 from the differential gradient (x 31, 185) . Arrows indicate dense cored vesicles ; asterisk indicates viral particles .

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gradient and fractions 2, 3, 4 of the differential gradient, no dense cored vesicles could be detected . Discussion The results of the differential centrifugation experiments reported show that the NA of the muse neuroblaetoma is present in a eedimentable form 01300 and ie therefore most likely associated with NA storage vesicles . These reeulte confirm and entend these of othRr workers (14) . The gradient centrifugation experiments allowed us to confirm the presence of NA in such vesicles and further demonstrated that these vesicles have a similar density as the "heavy" NA vesicles found in bovine sympathetic ganglia and fn bovine aad canine splenfc nerves . More recently such heavy vesicles have also been demonstrated in the dog spleen (27), cat spleen aad rat vas deferene (ï9), rabbit iris (28) and human nsuroblastoma (3, 4) and are thought to correspond to'the morphologically demonstrable large dense cored vesicles (10, 29). It is remarkable that the distribution of NA in the C mouse neuroblaetoma 13 00 upon equilibrium gradient centrifugation does not differ very much from the one of ß-glucuronidase and monoamine oxidase . This, of course, does not mean that NA is stored in lysosomes or mitochondria . It simply means that the equilibrium densities of these particles are very similar and that these particles cannot be separated under the present isopycaic centrifugation conditions . Indeed, the reeulte of the differential centrifugation (see Table I) show very clearly that the bulk of NA is not associated with lysosomes or mitochondria, since the distribution of NA completely differs from that of ß-glucuronidase and MAO which are used as marker enzymes for lyeosomes and mitochondria respectively . These results further suggest that differences in size amongst these organelles are to be expected rather than differences in density. A more important point, however, ie the fact that under the present experimental conditions no evidence was found for the occurrence of a second population of NA containing vesicles equilibrating at a low density, such as has been found for sympathetically innervated tissues . Previous electronmfcroecopic investigations on mouse neuroblastoma revealed tl~ presence of large dense cored vesicles, with a diameter of about 1500-2500 A and presumably containing catecholaminee (12) . Our own electronmicroscopie investigations on tissue sections con.~irm these data since large dense cored vesicles with a mean diameter of 1500 A were the only type of dense cored vesicles observed . These data, together with the results of the gradient studies, which show that only "heavy" vesicles are present, provide strong though indirect evidence for the view that the large dense cored vesicles might correspond to the "heavy" NA veeiclee . Electronmicroscopic investigation of fractions of the gradient, however, show that large dense cored vesicles arë only present in those fractions of the gradient where heavy veeiclee are found. These reeulte therefore directly support the view that also in moues neuroblaetoma, large dense cored veeiclee are the morphological correlate of the biochemically demonstrated heavy vesicles . Another point worth mentioning is the unusual distribution of DßH in the mouse neuroblaetoma which is completely different from that of peripheral adreaergic nerves where the majority of DßH appears to be contained within NA storage vesicles . Indeed, whereas a small part of the enzyme overlaps with the distribution of NA - a fact which might suggest that the "heavy" veeiclee contain DßH - by far the largest part of the enzyme ie found at a much lower

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density upon density gradient centrifugation where it almost completely parallels the distribution of alkaline phoephataee and '5-nucleotidase, i. e, plasma membrane marker enzymes, A similar distribution of DßH was also found iri some cases of human neuroblaetoma (4). Admittedly, it is well known that "light" NA storage particles - as found in adrenergically innervated tissues - also accumulate in this region of the gradient and therefore the possibility of the presence of DßH in much smaller particles was also considered . However, from the present experfrnents it is clear that no such small NA vesicles are present in G_1300 mouse neuroblaetoma nor that DßH occurs in small - eventually empty vesicles, We therefore conclude that the moat probable localization of the largest part of DßH is the plasma membrane . Referencea 1,

2. 3. 4. 5, 6, 7. 8. 9. 10 . 11, 12 . 13 . 14 . 15, 16, 17. 18, 19 . 20 . 21,

H. WINKLER and A . D, SMITH, Catecholamines (H, BLASCHKO and E, MUSCHOLL, ads. ) Handbook of Experimental Pharmacology, Vol. XXXIII, p, 900-933, Springer-Verlag, Berlin, Heidelberg, New York (1972) . L. B, PAGE and G, A . JACOBY, Medicine (Belt. ) 43 379-383 (1964) . H. HÔRTNAGL, H. WINKLER, H. ASOMER, H. F . FODLSCH and J. KLIMA, Lab, Invest . 2 7 613-61 q (1972), W, P, DE POTTER, A, F . DE SCHAEPDRYVER, F. DE SMET, M, J. DELBEKE and C . HOOFT, Experientia 30 1323-1324 (1974), I. W, CHUBB, W. P, DE POTTER and A, F. DÉSCHAEPDRYVER, Life Sci, _ll 323-333 (1972) . A. PHILIPPU, R . PFEIFFER, H, J. SCHifMANN and K. LICKFELD, Arch . Pharmakol, 258 251 -265 (1967), A, BURGER, A . PHILIPPU and H. J. SCHÜMANN, Arch, exp. Pathoh Pharmakol, _262 208-220 (1969) . W. P. DE POTTER, A. D, SMITH and A, F, DE SCHAEPDRYVER, Tissue & Cell 4 529-546 (1970), H. HÖRTNAGL, H. HbRTNAGL and H. WINKLER, J . Physiol. (Load. ) 205 103-114 (1969) . H, LAGERCR .ANTZ, Acta phyeiol, scand, , Suppl. 366 (1971) . Pr . AUGUSTE -T000O and Pr, SA TO, Proc . U. S. Nat. Acad. Sci, _64 311-315 (1969) . D . SCHUBERT, S. HUMPHREYS, G. BARONI and M. CORN, Proc . U. S . Nat, Acad, Sci. 64 316-323 (1969) . P. NELSON, W, RUFFNER and M. NIRENBERG, Proc . U, S, Nat . Acad . Sci. _64 1004-1010 (1969) . X. O. BREAKEFIELD, E. A. NEALE, J . H. NEALE and D. M. JACOBOWITZ, Brain Res, SZ 237-256 (1975) . Pr, AUGUSTE-TOCCO, G. H. SATO, I, CLAUDE and D, POTTER, Control Mechanisms in the E ression of Cellular Phenot s ,A, PADYKULA, ed, , pp, 109-120, Academic Press, New York (1970), N, B. OLMSTED, Ph, D. Dissertation, Yale University (1971), J . ROSS, J . B, OLMSTED and J. L. R06ENBAUM, Tissue &Cell 7 107136 (1975) . R, LAVERTY and K. M. TAYLOR, Anal . Biochem, 22 269-279 (1968) . R. GIANETTO and C. DE DUVE, Biochem. J. 5~ 433-438 (1955) . R, J . WURTMAN and J . AXELROD, Biochem, Pharmacol, _12 14391441 (1963) . C, DE DUVE, B. C. PRESSMAN, R . GIANETTO, R. WATTIAUX and F. APPELMANS, Biochem, J . 60 b04-617 (1955) .

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22, A, B, NOVIKOFF and M. HEUS, J . Biol . Chem . 238 710-716 (1963) . 23 . P. R . N . KIND and E. J. KING, J . Clin . Path. 7 322-326 (1954) . 24 . T. F. DIXON and M. PURDOM, J . Clin . Path . 7 341 -343 (1954) . 25 . O, H, LOWRY, J . Biol, Chem . 193 265-275 (1951), 26 . P, B, MOLINOFF, R. WEINSHILBOUM and J . AXELROD, J . Pharmacol. exp, Ther . _178 425-431 (1971) . 27 . W, DE POTTER, Peripheral Neurotransmission, p. 118, Arscia, Brussels (1976) . 28 . W, P . DE POTTER, F . DE SMET and E, CAMBIE, Arch, int. Pharmacodyn, _227 157-158 (1977), 29, M. A. BLSBY and M. FILLENZ, J . Physiol. (Loud. ) 215 163-179 ' (1971),