Subtypes of α1-adrenoceptors in hippocampus of pigs, guinea-pigs, calves and humans: regional differences

European Journal of Pharmacology - Molecular Pharmacology Section. 188 (1990) 9-16

9

Elsevier EJPMOL 90049

Subtypes of al-adrenoceptors in hippocampus of pigs, g~inea-pigs, calves and humans: regional differences Daniel H o y e r ~, C. Richard J o n e s 2 , , William F o r d ~ and J o s e M. Palacios ~ I Preclinical Research. Sandoz Ltd, CH-4002 Basel, Switzerland, and : Departmenl of Research, Unil'ersay Hospital. CH-4031 Basel, Switzerland

Received 22 May 1989, revisedMS received 17 August1989. accepted 29 August1989 Radioligand binding studies were performed with membranes of guinea-pig, pig. calf and human hippocampus using [~ZSl]BE 2254 (also known as [125I]HEAT) as the radioligand. [~2Sl]BE 2254 bound with similar high affinity to saturable populations of recognition sites in all four membrane preparations. Competition curves obtained with a variety of ligands (e.g., WB 4101, benoxathian, 5-methyl-urapidil) were biphasic and the profiles of the high- and low-affinity components of [12Sl]BE 2254 binding were similar in all four membrane preparations. The data suggest that [t2Sl]BE 2254 labels two subtypes of al-adrenoceptors in the hippocampus of these species. [3H]WB 4101 was used to label atA recognition sites in pig hippocampus membranes. [-aH]WB 4101 recognized with .high affinity an apparently homogeneous class of sites, as suggested by monophasic saturation and competition experiments. The rank order of affinity of the compounds for the high-affinity component of [~251]BE 2254 binding was similar to the rank order of affinity of these drugs for [3H]WB 4101 sites. The pharmacological profile of the low-affinity component of [t2Sl]BE 2254 binding was similar to that described recently for the ata-adrenoceptor cloned from DDT1 cells, In autoradiographic studies with human hippocampal slices, CEC (chloroethylclonidine). an alkylating agent described to shcrv:"selectivity for ata-adrenoceptors, dispk:ced preferentially [I~'SI]BE2254 binding from the molecular layer of the dentate gyms. In contrast, WB 4101 an alA-adrenoeeptor-selective ligand, displaced preferentially [t251]BE 2254 binding in the hilus and the CA3 region. The data show that 2 subtypes of al-adrenergic recognition sites can be identified in the hippocampus. In the humar, hippocampus, cqA sites are predominant in the hihis and the CA3 region. whereas ata sites are predominant in the molecular layer of the dentate gyrus. These subtypes show a similar pharmacological profile in man, pig, calf and guinea-pig, and may have a different functional role in these two areas of the hippocampus. ai-Adrenoceptor subtypes: [~251]HEAT([t~I]BE 2254): Hippocampus; (Guinea-pig): (Calf): (Human): (Pig)

i. Introduction There is increasing evidence for the heterogeneity of at-adrenoceptors but the topic remains controversial (see Flavahan and Vanhoutte, 1986; 1987; Docherty, 1987; McGrath and Wilson, 1988). a~-Adrenoceptor-mediated contraction in

Correspondence to: Daniel Hoyer, Preclinical Research, 386/525, Sandoz Ltd, CH-4002 Basel, Switzerland. * Present address: Merrell ResearchCentre, 16. rue d'Ankara, F-67000 Strasbourg,France.

smooth muscle is regulated differentially by extracellular calcium. In some tissues, the activation of inositol lipid turnover and the subsequent mobilisation of intracellular calcium suffice for the contractile effects whereas in other tissues, extracellular calcium plays an essential role in the receptor-induced contraction (Chin et al., 1987, for a review see Minneman, 1988). There is also e~4dence that ~t-adrenoceplor subtypes are pharmacologically different. In a recent series of publications, the groups of Minneman and Creese have expressed the view that subtypes of a~-adrenoceptors can be identified

0922-4106/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

10 both from radioligand binding studies and from functional studies (Morrow and Creese; 1986; Han et al., 1987; 1988). For instance, competition curves obtained with compounds such as WB 4101. benoxathian, and ARC 239 appeared to be shallow and could be resolved in a two-binding-site model when [12SI]BE 2254 or [3H]prazosin was used as radioligand. Chloroethylelonidine (CEC), an irreversible a:adrenoceptor antagonist, was shown to block preferentially a subpopulation of a:adrenoceptor binding sites, termed am, whereas the sites sensitive to WB 4101 were called a~A (Hart et al., 1987; 1988). Furthermore, it has been shown that [3H]WB 4101 labels a subset of the sites labelled by [3H]prazosin. These a~A sites are different from the other sites labelled by [3H]WB 4101, namely 5-HT1A sites (Norman et al., 1985a,b). Functional experiments carded out with peripheral rat tissues showed that some a|-adrenoceptor antagonists displayed different pA 2 values depending on the tissue investigated. These high and low pA 2 values were in agreement with highand low-affinity values derived from biphasic competition experiments. It was suggested that a subset of tz:adrenoceptors would stimulate contraction in smooth muscle via an increased phosphoinositide turnover, whereas another subset of a:adrenoceptors could cause contraction without an increased InsP3 production, in which case the entry of extraeellular calcium would play a major role. In addition to stimulation of inositol lipid turnover and activation of calcium channels, it has been suggested that ~q-adrenoceptors are also able to stimulate adenylate cyclase (Johnson and Minneman, 1986) and phospholipase A2. No obvious link has yet been found between the pharmacological characteristics of a receptor subtype and its activation of a given transduetion mechanism. There is no general agreement on the existence of a:adrenoceptor subtypes, and various explanations have been proposed to explain differences in affinity for both agonists (Coates et al., 1982; Bevan et al,, 1986; 1988) and antagonists (Hieble et al., 1986; Morrow and Creese, 1986; Han et al., 1987; 1988) for a:adrencceptors in different tissues. The situation is complicated by the fact that more than one subtype may be present in the

same tissue, e.g. vas deferens. The cq-adrenoceptor from the DDT1 hamster vas deferens cell line has, however, been cloned and expressed (Cotecchia et al., 1988), and it appears that the pharmacological characteristics of this receptor are of the a~B subtype. We have recently studied the distribution of a 1 recognition sites in the brain of various species (rat, guinea-pig, cat, monkey and man; see Palacios et al., t987), using [125I]BE 2254, a radioligand which labels a~a and a~B sites. Although the pharmacology of the sites was apparently the same m all species, their distribution was quite different. For instance, in comparison to the other species, human and monkey hippocampus showed a very high density of [lzSI]BE 2254 recognition sites. The aim of the present study was (1) to test whether subsets of ~:adrenoceptors could be identified in binding studies with pig, guinea-pig, calf and human hippocampus, and (2) to check whether WB 4101 (alA-selective) and CEC (aiBselective) could be used as tools for localizing subsets of a:adrenoceptors in hippocampal tissue sections.

2. Materials and methods

2.1. Radioligand binding Membranes from pig, guinea-pig, calf and human hippocampus were used for the radioligand binding studies. The membranes were prepared and experiments were carried out as described earlier (Engel and Hoyer, 1981; Hoyer, 1984; Pazos et at., 1984) with [12Sl]BE 2254 as the radioligand. Binding studies were performed with 50 #! radioligand (50°70 pmol/l in competition experiments), 50 ,ul buffer (Tris-HCl 10 mM, NaCI 154 mM, pH 7.5) or competing drug and 150 #1 of membrane preparations. Binding experiments were also carried out with [3H]WB 4101 and pig hippoc,ampal membranes. The membranes (750 ~1), were incubated with 200/tl buffer (CaCI 2 4 mM0 Tris-HCI 50 mM, pH 7.7) or competing ligand and 50/.d radioligand (1-2 nmol/l in competition experiments), using the same incubation conditions as

11 with [L~5I]BE 2254. Non-specific binding was defined in the presence of phentolamine 10 #M. Incubations were carried out at 3O°C for 30 rain and the assay was terminated by the standard rapid washing and filtration method. Competition and saturation experiments were analysed using " , - non-linear regression computer program. SCTFIT (De Lean, 1979; De Lean et al., 1980). The data are e~pressed as pK o values ( - l o g mol/1) and B ~ , values (fmol/mg of protein) ___S.E.M. Protein concentrations were determined by the method of Bradford (1976).

2.2. Autoradiography Autoradiographic studies were performed with slices from human hippocampus and rat brain. Humans brains were obtained at autopsy from five patients dying without a history of neurological disorders. The tissues were frozen on dry ice. Serial sections of hippocampus (10 ~m) were cut in a cryostat and mounted onto gelatin-coated slides. The sfides were stored at - 2 0 ° C until used. The experiments were carried out as described earlier (Patacios et al., 1987) with [~2~I]BE 2254 as the radioligand. Brain slices were brought to room temperature in 0.17 M Tris HCL pH 7.4 and incubated with [125I]BE 2254 (30-70 pM) and 150 mM NaCI for 2 h. The sections were then rinsed three times for 10 min in ice-cold Tris buffer. The sections were air-dried and exposed to [3H]ultrofilm (LKB, Sweden) for 12-16 h. Nonspecific binding was defined in the presence of prazosin 0.1 p.M. Binding was quantified densitometrically using Amersham 125I standards by means of an MCID image analysis system (Imaging Research Inc, St. Catherines, Ontario, Canada).

2.3. Drugs Ligands and drugs were from the following sources: prazosin (Pfizer, Sandwich, U.K.), 8OH-DPA L benoxathian and chloroethylclonidine (RBI, Wayland, MA, U.S.A.), phentolamine (Ciba-Geigy, Basel, Switzerland), 5-methy-urapidit (Byk-Gulden, Konstanz, F.R.G.), ARC 239 (Dr. Karl Thomae, Biberach, F.R.G.), BE 2254 (Beiersdoff, Hamburg, F.R.G.), yohimbine (Roth.

Frankfurt, F.R.G.). [t2~]BE 2254 was synthesized as described (Engel and Hoyer, 1981) or was from Amersham International (U.K.); [~H]WB 4101 was from DuPont NEN (Boston, MA, U.S.A.).

3. Results

3.1. Radio!igand bmd#tg [~"5I]BE 2254 labelled w;.tb high affinity a finite number of sites in hippocampal membranes of guinea-pig, pig. calf and man (see table I ). Experiments were performed with various co~pounds known to interact with eq-adrenoceptors. Figure 1 shows the [ ~ I ] B E 2254 binding in human and pig hippocampus: [1251]BE 2254 labelled two different recognition sites. Most of the compounds tested showed biphasic competition curves, although this was not obvious for compounds such as prazosin, BE 2254 or phentolamine, and overall, the compounds showed only small selectivity. The high-affinity component (40-60% of specific binding) showed affinity values in the 1-10 nmol/l range for WB 410t, BE 2254, ARC 239, benoxatlfian and in the 10-I00 nmot/I range for phentolamine. yohimbine and 5-methyl-urapidil. The rank orders of affinity of the drugs for either the high-afFinity or the low-affinity component were similar in all four speck.~. As already reported (Gross et al_

TABLE 1 Results from ..~,~turationexperiments~ith [~z~I]BE2254 and lsH]WB 4101 ia hippocamp~l membranes. Membraneswere incubated as de~ribed iB Mate"ialsaad methodswilh [a:.~IIBE 2254 00-80 ~.g/assay) or ['~HiWB 4101 (600.900 #g/assay). B~, values are expres.~d as fmol/mg protein±S.E.M_ affinity values a~ pKD (--log tool/l) ±S.E.M. of rl experirnenls. Membrane

Ligand

B~

campus Calf hipp~campus Pig hippocampus Pig hippocampus

[Iz-~I]BE2254364 ±80

pKD

n

10~02±0.12 4

[~'2~I]BE22541"~.~.i±11.2 t0.38±0.05 5 [~'~SilBE2254t31.0_+ g.1 t0.25_+0.03 5 [~H]WB410t 2I.$± 1.4 9.75±0.04 4

12 TABLE 2 Affinity values of various compounds for []~I]BE 2254 and [3H]WB 4101 sites in hippocampus membranes. The affinity values of the tested compounds are expressed as pK o values ( - log tool/l) 4-S.E.M. of n = 3-~ values• High- and low-affinity values are listed where a biphasie figwas significantly better than, a monophasic fit: [12Sl]aE 2254 binding (a). In case of [3H]WB 4101 binding (b) the competition experiment:: were an~-y=-edfor a o.':e-site model. Drug

WB4101 BE2254 Prazosin Yohimbine Phentolamine ARC239 Benoxathian 5-CH3-urapidil

Human ~ [IZSI]BE 2254

Gninea-pig ~ [125I]EE 2254

Pig ~ [1z~I]BE 2254

Calf ~ [ t25I]BE 2254

pK D high pK Dlow

pKD high pK o low

pKD high

pKD high pKt> low

8.834-0.137.282:0.18 9.024-0.147.43+0.23 9.674-0.127,234-0.40 6.914-0.M 6.19+0.31 7.81+_+_0A6 8,382:0.27 6.28_+0.18 8.48_+0.32 7,464-0.19 8,484-0.26 6,474-0.24

8.354-0.10 8.67+0.25 9.972:0.08 6,814-0.11 8.234-020 8.18_+0.15 8.514-0.10

1988), 5-methyl-urapidil, a l t h o u g h not the m o s t ~9otent, was the c o m p o u n d which s h o w e d the highest selectivity for the high-affinity sites. The affinities of the c o m p o u n d s are listed in table 2. Pig Hip[mc~mp~

50

Human Hipl~ca rnl.,J~

50-

O-

0

pKD tow

7.494-0.19 9.524-0.18 7.964-0.37 9.16+0.15 7.454-0.02 9.53.+_0.14 7.914-0.22 9.07_+.0.24 6.874-0.37 10.104-0.15 7.004-0,32 9A14-0.15 7.822:0.27 6.77+0.43 8.04+0.17 8.384-0.28 6.954-0.14 8.304-0.10 6.624-0.10 8.944-0.18 7.704-0.62 8.704-0.17 7,304-0.10 8.344-0.47 7.274-0.25 8.634-0.28 8.92+0.19 6.474-0.25 7.834-0.19

1

=-100-

7.36+0.14 7.65+0.21 0.63+0.10 6.794-0.14 6.93+0.23 6.80+0.50 7.204-0.30 5.104-0.20

Pig b [ "~HIWB 4101 pKD 9.054-0.5 9.154-0.05 9.00+0.10 7.394-0.18 7.554-0.07 8.67__+0.22 8.764-0.02 8.80:~__0.22

[3H]WB 4101 b i n d i n g w a s studied in pig hipp o c a m p a i m e m b r a n e s . B i n d i n g was of high affinity a n d s a t u r a b l e (table 1). T h e c o m p e t i t i o n curves o b t a i n e d w i t h the tested c o m p o u n d s were essentially m o n o p h a s i c s u g g e s t i n g that [3H]WB 4101 labels a single p o p u l a t i o n o f sites. B i n d i n g to 5-HTtA sites c o u l d b e ruled out since 8 - O H - D P A T , a selective a n d high affinity 5-HTtA receptor agonist, s h o w e d very low affinity for the sites labelled b y [3HIWB 4101 ( p K o < 5). The r a n k o r d e r of affinity of the drugs tested for [3H]WB 4101 b i n d i n g was s i m i l a r to that observed for the high-affinity c o m p o n e n t of [i-'5I]B E 2254 b i n d i n g ( t a b l e 2). Or~ the o t h e r hand° the low-affinity c o m p o n e n t o f [t251]BE 2254 b i n d i n g has a profile s i m i l a r to that described for a n a m site ( H a n et al., 1987; 1988; C o t e c c h i a et al., !988).

3.2. Autoradiographic studies

500......

~'lb'

/~ ' b ' , t

• ~ Inrugl 4moVh

Fig. I. Experiments on core~tition for [t2SlIBE 2254 binding sites in pig and human hippocampal membranes. Competition curves of yohimbine (lop). benoxathian (middle) and WB 4101 (bottom) for II:51IBE 2254 binding in pig (left panel) and human hippocampns (right panel). The ~ specific binding is plotted vs. concentrations of the drug ( - log tool/t).

In a p r e l i m i n a r y set of experiments, h u m a n h i p p o c a m p a l slices were i n c u b a t e d ira the presence of i n c r e a s i n g c o n ~ n t r a t i o n s o i W B 4101. A n a l y s i s of the c o m p e t i t i o n curves o b t a i n e d for the different regions of the h i p p o c a m p u s gave a Hill slope not different from unity for ti:e hilus (1.06 + 0.11), c o n s i s t e n t with single site with high affinity ( p K D 8.61), whereas the i n h i b i t i o n curve for the molecular layer of the d e n t a t e g y r u s was shallow (slope = 0.63 + 0.09) a n d the d a t a were significantly better

B ::i::;

............

<

E

F •

2

;-/

i.'

N2.

CEC

1 0 0 IJM

Fig. 2. Autoradiographic visualization of aFadrenarg': receptors in the human ~Jppc~:a~pa~ :,~rma~i~n~ The fi.gure shc,ws phcr~ographs from autoradiogrm'as performed with [~:51]BE 92~,4 in the denta~ gy-a~ ~,nd CA. ~'eg~o~. h ~ de~,ies of b[ndtn~ "~,ere observed in the molecular layer of the dentate gyrus (Mo~), in lhe hilus (H) and CA3 etre:~, tn co~ras~, ~o~, ~eve~sof binding were observed in the granule cell layer (Gr). T~I~ binding ~A non-specific binding (D). WB alOt, 3 and t(~ nM (B-C). pretreatmen~ with CEC 16 arid 100 nV/(E-F), Bar = 5 mmo

fitted b y a two-site analysis. T h e h i g h - a f f i n i t y site h a d a p K n o f 8.57 a n d r e p r e s e n t e d 28% o f the b i n d i n g ; t h e p K o o f the l o w a f f i n i t y w a s 6~98 a n d r e p r e s e n t e d 72% o f the b i n d i n g .

Brair, slices were i n c u b a t e d und~:r o n e o f three sets o f c o n d i t i o n s : i n c u b a t i o n s b u f f e r a l o n e o r wi~h the a d d i t i o n o f e i t h e r 10 o r 100 t t M C E C f o r 10 m i x a n d t h e n w e r e w a s h e d extensively. Subse-

14

quently, the slices were incubated in the absence (total binding) or in the presence of 0.1 t~M prazosin (non-specific binding) or 3, 10 and 30 n M WB 4101. These concentrations were chosen in order to displace either high-affinity (a~A) binding alone or both high- and low-affinity binding (a~A + ~xm). AT

ZB

7~ ~

T

t

Fig. 3. Bar graph repres.~ntation of quantitative autoradio-

graphic experimentsperformed with human hippocampal forma~.~.. (AI Effects of WB 4101 (3. 10 and 30 aM, in the absence of CEC pretrealment) on [ix~I]BE 2254 binding in human hippocampus(molecularlayer ~f the dentate gyrus and hihis). (BI Effects of CEC pretreatment on [iz~IIBE 2254 binding in human hippocampus.(C) Effects of WB 4101 on [~2~IIBE 2254 binding in human hippocampus after a 10 pM CEC pretreatment. The data represent specific binding expressed as % of controls (absence of drug or treatme,~t) S.E.M. of three determinations.

Figure 2 shows the [125I]BE 2254 binding in the h u m a n hippocampus under the conditions described above. [t25I]BE 2254 labelled a high density of sites in the h u m a n hippocampus, particularly in the dentate gyrus and the CA3 region. In t i e dentate g3,a'us, the molecular layer was very rich in binding sites. High densities of binding were also seen in the subgranular area and in tile hilus, while the granular cell layer showed a low density of binding sites. P r ~ o s i n (0.1 /tM) was able to displace most of this bii,ding. Increasing concentrations of WB 4101 displaced concentration-dependently the binding from the hilar region and CA3 area, whereas the binding in the molecular layer of the dentate gyrus was displaced at higher concentrations only. Opposite effects were observed when the slices were preineubated with CEC, then washed (CEC is an alkylating agent). Whereas CEC displaced in binding from the molecular layer of the dentate gyrus at 10 ttM and even more at 1 0 0 / t M , [I~5I]BE 225a binding was much less affected in the hilar regions. This is further illustrated in fig. 3 which shows that, without CEC pretreatment, WB 4101 produced effects opposite to those observed with CEC. It can also be seen that the binding remaining after CEC treatment was readily displaced by WB 4101 in both the hilus a n d the molecular layer of the dentate gyrus. It is clear that WB 4101 displaced more binding in the hilus than in the molecular laver of the dentate gyms. These data suggest that a i a and ain sites are present in both structures but that the hilar regions contain predominantly ~xta sites and the molecular layer of the dentate gyrus contains predominantly a m sites. There was no evidence for similar effects in the rat brain, where [125I]BE 2254 binding was much less pronounced, and no regional differences in the distribution could be shown for CEC- and WB 4101-sensitive binding (data not shown).

4. Discussion The data suggest that two populations of pharmacologically different a~-adrenoceptors, namely WB 4101-sensitive sites (termed atA ) and CEC-sensitive sites (atB), are present in the pig,

guinea pig, calf and human hippocampus. The pharmacolo~, of the two sites is very similar and apparently sh~,ws no species differences. Thus, the high-affinity component of the sites labelled with [125I]BE 2254 has a pharmacological profile very similar to that of the sites labelled with [3H]WB 4101, suggesting that these sites are of the a~A type. Hart et al. (1987; 1988) have described these sites in the hippocampus and vas deferens, where they appear to be linked to activation of Ca 2+ channels. On the other hand, the low affinity component of the sites labelled with [~25I]BE 2254, is very similar to the profile of the sites termed eqB by Han et al. (1987; 1988). These sites have been described in the liver and the spleen, where they appear to be linked to activation of inositol phosphate metabolism and mobilisation of intracellular calcium. The eqB-adrenoceptor has recently be identified, cloned and expressed from DDT1 ceils, a hamster vas deferens cell line (Cotecchia et al., 1988). However, both these receptors are also present in hippocampus and rat vas deferens, where inositol phosphate metabolism has also been described (Fox et al., 1985; l~,finneman et al., 1987). It could be argued that the site showing low affinity for prazosin is not related to aradrenoceptors. However, one should keep in mind that the radioligand concentration was very low (about 50-70 pmol/1). It would be surprising that the radiofigand was labelling a site not related to a~-adrenergic sites at such low concentrations, although, this possibility cannot yet be completely ruled out. Furthermore, recent data from functional and second messenger studies reported by Beckeringh and Brodde (1989), suggest an a~adrenoceptor population which is antagonized by prazosin with pA 2 values in the range 7.6-8.0, that is close to the values we now found. Also, the a~-adrenoceptor cloned and expressed by Cotecchia et al. (1988), has a rather low affinity for prazosin, WB 4101, phentolamine and yohimbine (pK ~ values ,~f 8.6, 8.0, 6.5 and 5.6, respectively). On the other hand, the subtypes described by Morrow and Creese (1986), indeed show a very high affinity for prazosin, suggesting that more than two subtypes of a~-adrenoceptors may exist. The results obtained from radioligand binding studies are supported by the results of autoradio-

graphic studies on human hippocampal slices. There are remarkable differences in the distribution of WB 4t01- and CEC-sensitive sites. WB 4101 displaces preferentially [1251]BE 2254 binding from the hilus and CA3 region, whereas the opposite occurs with CEC. This compound selectively displaces the binding from the molecular layer of the dentate gyms. These data are consistent with the competition curves obtained for the two areas. According to the relative affinities of CEC and WB 4101 for the two subtypes it is proposed that the cq-adrenoceptors in the hilus are predominandy of the atA type, whereas the receptors in the molecular layer of the dentate gyrus are predominantly of the ~xlB type, which explains the greater displacement of the radioligand by CEC in this layer. The effects occur at concentrations which are consistent with the affinity values reported toy Hart et al. (1987; 1988). As reported from an earlier study, the major difference between primates and non-primates is the very high concentration of al-adrenergic sites in the hippocampus of primates. This was one of the reasons for performing the present study. It was expected that, due to these high concentrations, a ~ossible difference in regional distribution of ~ladreaoceptor subtypes might be detected in primate hippocampus. This was indeed the case. In contrast we could not show a clear differential distribution of vabtypes of a~ recognition sites in rat hippocampus, perhaps because the level of binding was too low to permit a proper characterization of the distribution of subtypes of al-adrenoceptor recognition sites. In addition to showing the presence of subtypes of at-adrenergic sites in the hippocampus, the present data support the view that ~x~-adrenoceptors in the hilus and the molecular layer of the dentate gyrus of the human hippocampus have different functions which remain to be explored.

Acknowledgements We are grateful to Dr. A. Probst (Departmentof Neuropathology, lnsdtme of PatholoE~', University of Basel) for his help in obtainip~ghuman brain samples,and to Dr. J.R. Fozard for critical readingof the manu~ript.

16

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