Comparison of cholecystokinin metabolism by membrane preparations from the human astrocytoma clone D384 and the neuroblastoma line SH-SY5Y

~)

Pergamon

0197-0186(93)E0007-3

Neurochem. Int. Vol. 24, No. 4, pp. 369- 377~ 1994 Copyright C 1994 Elsevier Science Lid Printed in Great Britain. All rights reserved 0197 0186/94 $7.00+1).00

C O M P A R I S O N OF C H O L E C Y S T O K I N I N METABOLISM BY M E M B R A N E P R E P A R A T I O N S FROM THE H U M A N A S T R O C Y T O M A CLONE D384 A N D THE N E U R O B L A S T O M A LINE SH-SY5Y MARGARET DOS SANTOS MEDEIROS* a n d ANTHONY J. TURNER Department of Biochcmistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, U.K. (Received 28 September 1993 ," accepted 5 November 1993)

Abstract

Both the sulphated and non-sulphated forms of cholecystokinin (CCK) octapeptide are susceptible to hydrolysis by the cell-surface peptidases endopeptidase-24.11 (NEP), angiotensin converting enzyme and aminopeptidase N (AP-N). Indirect studies have previously implicated an elastase-like serine cndopeptidase in CCK metabolism in brain. Wc have therefore compared the hydrolysis of CCK, in both sulphated and non-sulphatcd R~rms by solubilized membrane preparations from the human astrocytoma clone D384 and the neuroblastoma line SH-SY5Y. Selective peptidase inhibitors were used to elucidate the principal activities involved in CCK metabolism. In the glial cell line the hydrolysis of cholecystokinin octapeptide (CCK-8), sulphated or non-sulphated, was inhibited predominantly by the NEP inhibitor, phosphoramidon (PR). In contrast, in the neuroblastoma line, angiotensin converting enzyme (ACE) was seen to play a major role in metabolism of CCK-8 with a lesser effect attributable to NEP but with some differences between sulphated and non-sulphated forms reflecting the preference of ACE for CCK-8ns. In neither cell line was a significant effect of the serine peptidase inhibitor Dip-F seen on CCK metabolism arguing against the presence of a putative CCK-degrading serine peptidase in these cell lines. Both NEP and ACE remain as candidates for inactivation of CCK at the cell surface.

Cholecystokinin ( C C K ) is a regulatory peptide which exists in several molecular forms varying in length from 4 to 58 a m i n o acid residues (Rehfeld, 1978; C a n t o r a n d Rehfeld, 1987: C a n t o r , 1989). All these peptides share the c a r b o x y - t e r m i n a l region, displaying different lengths o f their a m i n o - t e r m i n a l extension. The c a r b o x y - t e r m i n a l octapeptide (CCK-8) is comm o n to most m e m b e r s of the C C K / g a s t r i n family and retains the whole biological activity. Carboxyterminal a m i d a t i o n s and sulphation are essential for C C K function (Vinayek et al., 1987; H u a n g et al., 1989). C C K is found in the l cells of intestinal m u c o s a with higher c o n c e n t r a t i o n s in d u o d e n u m and upper *Author to whom all correspondence should be addressed. Ahbreciations: ACE, angiotensin converting enzyme; AM, amastatin: AP-N, aminopeptidase N; CCK-8, cholecystokinin octapeptide: CCK-8s, sulphated l\~rm of CCK-8 ; CCK-Sns, non-sulphated form ofCCK-8 : DipF. diisopropyl fluorophosphate; DPP-IV, dipeptidylpeptidase IV, MK 422, enalaprilat: NEP, neutral endopeptidase (endopeptidase-24. I I ) ; PR, phosphoramidon.

j e j u n u m (Buffa et al., 1976) a n d C C K immuno-reactive nerve fibres have been d e m o n s t r a t e d in the pancreas of several species (Larsson, 1979 : Rehfeld et al., 1980). In plasma, after a meal, the most a b u n d a n t circulating C C K molecules are the large forms such as CCK-58, CCK-39, CCK-33, C C K - 2 2 a n d also C C K - 8 (Liddle et al., 1985). However, in the central nervous system, short fiagments of the peptide like C C K - 4 and C C K - 8 sulphated (CCK-8s) and non-sulphated (CCK-Sns) are p r e d o m i n a n t (Dockray, 1982; Dockray et al., 1985 ; Rehfeld et al., 1985). A variety of physiological roles are attributable to C C K as a gastrointestinal h o r m o n e a n d / o r neurot r a n s m i t t e r (Lewis and Williams, 1990). These include : stimulation of pancreatic enzyme secretion : inhibition of gastric emptying ; p o t e n t i a t i o n of a m i n o acid-induced insulin secretion in h u m a n s ; and direct stimulation of insulin secretion by activation of pancreatic/~-cell C C K receptors, regulation of gall bladder c o n t r a c t i o n and food intake. In the C N S , C C K can produce antinociccption (Hill et al., 1987). C C K receptors are k n o w n to exist in at least two distinct subtypes, classified according to their atiinity 369

370

MARGARET DOS SANTOS MEDEIROS and ANTHONY J. TURNER

for the sulphated a n d n o n - s u l p h a t e d forms o f C C K 8 a n d the n o n - p e p t i d e C C K a n t a g o n i s t Devazepide (MK-329) (Innis a n d Snyder, 1980 ; D o u r i s h a n d Hill, 1987; E v a n s et al., 1986). C C K - A receptors are pred o m i n a n t l y f o u n d in peripheral tissues but are also seen in primate central nervous system, displaying high affinity for C C K - 8 s a n d MK-329, a n d low affinity for C C K - 8 n s and CCK-4. C C K - B receptors are the classical brain receptors which show poor disc r i m i n a t i o n between sulphated and n o n - s u l p h a t e d forms, and high affinity for the antagonist L365,260 (Lotti a n d Chang, 1989). Both the C C K - A a n d C C K B type of receptors have recently been cloned ( W a n k et al., 1992a,b) and their seven putative transm e m b r a n e d o m a i n s are characteristic of the G protein-coupled receptor superfamily. There is controversy in the literature concerning the enzymes involved in the metabolic disposition of CCK. M a t s a s et al. (1984) have shown that endopeptidase-24.11 ( N E P ; neutral e n d o p e p t i d a s e : EC 3.4.24.11) can hydrolyse C C K - 8 s in vitro at two distinct sites. G l y 4 - - T r p ~ a n d AspT--Phe~NH> The profile of hydrolysis of C C K - 8 s by pig brain striatal synaptic m e m b r a n e s c o r r o b o r a t e d these results, since hydrolysis was suppressed in the presence of the N E P inhibitor p h o s p h o r a m i d o n (PR). Evidence for a contribution from an unidentified a m i n o p e p t i d a s e was also presented ( M a t s a s et al., 1984). Angiotensin converting enzyme ( A C E ; EC 3.4.15.1) can also hydrolyse C C K in t,itro (Dubrcuil et al., 1989) at the penultimate peptide b o n d , and as a secondary cleavage, A C E subsequently releases di- or tri-peptides from the c a r b o x y - t e r m i n a l end o f the remaining aminoterminal fragments. A t t e m p t s to examine the effects of peptidase inhibitors on the recovery o f C C K released from brain slices have, however, produced equivocal results. Zuzel et al. (1985) reported that t h i o r p h a n ( N E P inhibitor) did not significantly affect the recovery o f the peptide from slices of cerebral cortex and had only a small effect in the case o f striatal slices. Subsequent reports (Rose et al., 1988; 1989; C a m u s et al., 1989) have shown that serine peptidase inhibitors sucl~ as diisopropyl f l u o r o p h o s p h a t e (Dip-F) could potentiate the recovery of released CCK. These studies implicated a novel elastasc-like serine e n d o p e p t i d a s e in brain that cleaves the two peptide b o n d s of C C K - 8 where the carboxyl g r o u p is d o n a t e d by a m e t h i o n i n e residue. In order to explore the potential roles of metallopeptidases a n d serine peptidases in C C K hydrolysis we have therefore c o m p a r e d the metabolism of C C K 8 sulphated and n o n - s u l p h a t e d by m e m b r a n e fractions from two h u m a n cell lines, one of n e u r o n a l

(SH-SY5Y) a n d one of glial (D384) origin. Selective peptidase inhibitors have been used to identify the principal enzymes involved. EXPERIMENTAl. PROCEDURES

Materials

CCK-8s (26 33) and CCK-8ns (26 33) were purchased from BACHEM UK Ltd, Essex, England. PR was acquired from the Protein Research Foundation, Osaka, Japan. Amastatin (AM), Dip-F and bicinchoninic acid (BCA) solution were from Sigma Chemical Co., Dorset, England. Enalaprilat (MK 422) was a gift from Merck, Sharpe & Dohme, N J, U.S.A. NEP, aminopeptidase N (AP-N : EC 3.4.11.2) (AP-N) and ACE were purified from pig kidney cortex as described previously (Fulcher and Kenny, 1983: Hooper and Turner, 1987) and their homogeneity confirmed by SDS-polyacrylamidc gel electrophorcsis. AP-N purification follows the same procedure applied for NEP except that GK8C 1 was used instead of GK7C2 for lhe immunoaffinity chromotography step. Cell cuhure and membrane preparation

The cultures of D384 and SH-SY5Y ceils were generously provided by Dr A. J. Balmforth and Dr P. F. T. Vaughan. D384 cells were maintained in culture as described previously (Balmforth et al., 1986 ; 1988) plated in 175 cm" tissue culture flasks and used at confluence one week later (Lyall et al., 1988). The cells were washed briefly with 5 mM Tris HCI buffer, pH 7.4, at 4 C and disrupted by freeze thawing in methanol/dry ice t\)r 1 rain. After scraping the cells from the flasks into 5 mM Tris HC1 buffer, pH 7.4, they were sedimented by cent rifugation at 1000 g for 10 rain and resuspended in 50 mM HEPES buffer, pH 7.5 containing 20 mM MgCI2. The cells were homogenized by using N~ cavitation and centrifuged at 1000g for 10 rain at 4 C. The supernatant was centrifuged at 100,000 g, for 1 h at 4 C and the pellet resuspended in 1 m150 mM HEPES buffer pH 7.5, containing 60 mM octyl glucoside. The detergent solubilized pellet was incubated at 4 C for I h and centrifuged at 12,000g for 5 rain to remove particulate material. The solubilized membrane fi'action was stored at 20 C until required. The preparation of a solubilized membrane fraction from SH-SY5Y cells, cultured as described by Vaughan and Ball (1990), used the same procedure except that the freeze thawing step was unnecessary to lyse the cells and was omiued. C C K metabolism

CCK-Ss and CCK-Sns (50 ,uM) were incubated separately with NEP (100 ng protein/30 rain), ACE (100 ng protein/4 h, 1 h), AP-N (100 ng protein/4 h, 1 h), D384 cells (50 Hg protein/6 h) and SH-SY5Y cells (114 fig protein/6 h) in the presence or absence of specific inhibitors in 0.1 M Tris HCI buffer, pH 7.4 in a total volume of 100/d at 37 C. For assay with ACE, 0. I M Tris HC1 buffer, pH 8.3, containing 0.3 M NaCI, and 10 I~M ZnCI~ was used. The products of hydrolysis of CCK-Ss and CCK-Sns were analysed by reverse-phase HPLC by using a HBondapak C~s column as described elsewhere (Matsas et al., 1984). The fractionation used a linear gradient ofacetonitrile from 4.8 60% in 0.08% H~PO4, pH 2.5, followed by 5 rain isocratic elution in final conditions. The products were monitored at 214 nm.

371

Cholecystokinin metabolism in cell lines

Protein e.s'limalion Protein concentrations were determined by the BCA method (Smith et al., 1985) using bovine serum albumin as standard. R ES U LTS

Metabolism q f CCK-8s and CCK-8ns by purified membrane peptidases CCK-8s and CCK-Sns were incubated individually with NEP, ACE and AP-N and their products of hydrolysis were analysed by reverse-phase HPLC (Figs 1 and 2). By comparing their hydrolytic rates, CCK-Sns was adjudged to be more efficiently hydrolysed by ACE and AP-N than was CCK-8s (Figs 1 and 2). Such a significant difference in hydrolysis rate was not observed when incubating both peptides with NEP. Figure 3 shows the primary structure of CCK8s and tile hydrolysis sites for NEP, AP-N, ACE and the putative serine endopeptidase. NEP cleaves CCK8s at AspT-PheSNH~ and Gly4-Trp 5. The putative scrine endopeptidase is reported to hydrolyse CCK-8s at Met3-Gly~. The major hydrolysis site for ACE is at Me('-Asp 7 whereas for AP-N is at AspLTyr-'(SO3H). Prolonged incubation with AP-N and ACE produces

further degradation. With 100 ng NEP the half life for hydrolysis of CCK-8s was 17.4 rain compared with 19 min for the hydrolysis of [DAla-'-LeuS]-enkephalin, the synthetic peptide used in routine assays of NEP.

Hydrolysis o1' CCK-8s by D384 and SH-S }I5 Y ('ell membranes CCK-8s was degraded by a solubilized membrane preparation from D384 cells (50 ttg protein) [Fig,4(a,b,c.)] and its hydrolysis was completely inhibited by PR, all product peaks being eliminated in the presence of the inhibitor. The serine proteinase inhibitor, Dip-F, [Fig. 4(c)] and the aminopeptidase inhibitor, AM, had virtually no effect on the production of any of the CCK-Ss metabolites. The concentration and time of incubation with Dip-F were sufficient in parallel experiments, to inhibit fully the serine peptidase, dipeptidyl peptidase IV (DPP-IV). The inhibition of production of individual product peaks is shown in more detail in Table 1. When incubated with a solubilized preparation of SH-SY5Y cell membranes (115 ILg protein) a more complex pattern of metabolism was seen [Fig.4(d)] : the hydrolysis of CCK-8s was predominantly

CCK-Ss

CCK-Ss i

CCK-Ss

b.

C.

1

A2~

i

i

i

TIME (rain)

Fig. 1. HPLC analysis of CCK-8s (50 /tM) metabolism by NEP. ACE and AP-N. CCK-8s (50 ltM) was incubated with peptidases at 37C. Products were resolved by HPLC as described in Experimental Procedures. Metabolism of CCK-8s is shown by: (a) NEP (100 ng protein, 30 min, hydrolysis - 86%) ; (b) ACE ( I00 ng protein protein, 4 h, hydrolysis - 19.8%) ; (c) AP-N (100 ng protein, 4 h, hydrolysis 11.1%).

372

MARGARET DOS SANTOS MEI)EIROS and ANTHONY J. TURNER Q.

b.

CCK-Ons

C.

CCK-Sns

I

A21A

C(~K-Sns

J I

I

I

I

I

5

10

15

20

0

|

5

I

I

l

I

I

I

I

I

10

15

20

0

5

10

15

2O

TIME (mini Fig. 2. HPLC analysis of CCK-Sns (50 I~M) metabolism by NEP, ACE and AP-N. CCK-Sns (50 I~M) was incubated with peptidases at 37 C. Products were resolved by HPLC as described in Experimental Procedures. Metabolism of CCK-8ns is shown by (at NEP (100 ng protein, 30 min, hydrolysis - 88.7%); (b) ACE (100 ng protein, Ih, hydrolysis - 55.4%): (c) AP-N (100 ng protein, 1 h, hydrolysis 66.7'!;).

1

2

3

4

3

Asp-Tyr-[SO3H] Met Gly Trp-Met-Asp-PheNH2 Fig. 3. Hydrolysis sites of CCK-Ss by peptidascs. The observed cleavage site is indicated by an arrow between the two amino acids. Numbers correspond to: (11 AP-N: (2) putative serinc endopeptidase: (3) NEP: (4) ACE. inhibited by PR and the A C E inhibitor, MK-422, whereas D i p - F and A M had little or no inhibitory effect (Table 2).

14ydrolysis of CCK-8ns by D384 and S H - S Y 5 Y cell membranes The d e g r a d a t i o n of C C K - S n s by D384 solubilized cell m e m b r a n e s (50 ILg protein) [Fig. 5(d)] follows the same profile observed in this cell line ['or CCK-8s, with p h o s p h o r a m i d o n being the m a j o r inhibitor and again Dip-F, A M and M K - 4 2 2 show very little suppressive effect on the f o r m a t i o n of C C K - 8 n s metabolites (Table 3). This would be consistent with a m a j o r role of N E P in C C K metabolism in this cell line.

The incubation of CCK-Sns with SH-SY5Y solubilizcd cell m e m b r a n e s (114 fig) showed more extensive hydrolysis which was almost entirely inhibited by M K - 4 2 2 and negligibly affected by other inhibitors such as Dip-F, and A M . PR had only a partial inhibitory action [Fig. 5(a,b,c): Table 4]. These findings would indicate A C E as a key enzyme in the metabolism of C C K in SH-SY5Y cells. DISCUSSION Several cell-surface peptidase activities have been implicated in the degradation of the a b u n d a n t neuropeptide C C K (Matsas e t a / . . 1984: Dubreuil e t a / . ,

Fig. 4. (opposite) HPLC analysis of CCK-8s (50 #M) metabolism by D384 and SH-SY5Y cell membrane preparations. CCK-8s (50/*M) was incubated with D384 and SH-SY5Y membrane preparations for 6 h at 37 C. Products were resolved by HPLC as described in Experimental Procedures. Metabolism of CCK8s is shown by: (a) D384 cell membranes (50/*g protein, hydrolysis = 10.2%); (b) D384 cell membranes (50 /xg protein, hydrolysis = 0 % ) + 1 0 pM PR; (c) D384 cell membranes (50 /~g protein, hydrolysis = 5.6%)+ 1 mM Dip-F; (d) SH-SY5Y cell membranes (114 pg protein, hydrolysis = 13.8%)

373

Cholecystokininmetabolismin cell lines CCK-Ss

CCK-Gs

C.

Q.

1

A21a

i

I

5

10

2~

CCK'Os

CCK-6s

b.

d.

1

A214 PI~

~. k_.

j

k_... 2~

i

15 TIME ( m i n ) Fig. 4~ legend opposite.

374

MARGARET DOS SANTOS MEDEIROS a n d ANTHONY J. TURNER

Table I. Effectof inhibitors on metabolismof CCK-8s by solubilizedpreparations of D384 cell membranes Product elution time (min)

Inhibition (%) 10 l~M PR

10/~M MK 422

. 10/~M AM

4.62 7.90 9.58 13.18

100 100 100 100

32.7 9.7 0 0

0 0 0 (1

.

. . I mM Dip-F 19.3 6.0 0 0

CCK-8s (50 #M) was incubated with 50 #g protein of D384 cell membranes for 6h (hydrolysis= 10.2%) and in the presence of inhibitors. The products were analysed by reverse-phaseHPLC as described in Experimental Procedures. Table 2. Effect of inhibitors on metabolism of CCK-Ss by solubilized preparations of SH-SY5Y cell membranes

Product elution time (min)

Inhibition (%) 10 I~M PR

10 ILM MK 422

10 I~M AM

10.48 13.04 13.22 13.80

82.0 100 100 24.0

100 0 0 69.2

18.7 15.9 11.4 13.4

I mM Dip-F 41.7 0 11.3 19.3

CCK-Ss (50 #M) was incubated with 114 #g protein of SH-SY5Y cell membranesfor 6h (hydrolysis= 13.8%) and in the presenceof inhibitors. The products were analysed by reverse-phaseHPLC as describedin Experimental Procedures. Table 3. Effectof inhibitors on metabolismof CCK-8ns by solubilizedpreparationsof D384 cell membranes Product elution time (rain)

Inhibition (%) 10 ,uM PR

10 ,uM MK 422

10/~M AM

I mM I)ip-F

7.58 12.86 13.24 13.98

100 100 100 100

72.8 14.0 9.7 0

18.1 13.4 16.0 9.1

20.4 19.0 13,7 8.8

CCK-8ns (50 ,uM) was incubated with 50 #g protein of D384 cell membranesfor 6h (hydrolysis= 14.7%) and in the presence of inhibitors. The products were analysed by reverse-phaseHPLC as described in Experimental Procedures.

1989; C a m u s et al., 1989). O f these candidates only N E P has been shown by a variety o f criteria to fulfil a general role as a " n e u r o p e p t i d a s e " , especially in the metabolism o f opioid peptides and tachykinins as well as natriuretic peptides. It has previously been shown that, in synaptic m e m b r a n e preparations, the N E P inhibitor P R blocks C C K degradation (Matsas et al., 1984). Some inhibition o f C C K metabolism was also effected by an a m i n o p e p t i d a s e inhibitor although the a m i n o p e p t i d a s c itself was not identified in that study. The N-terminal aspartyl residue o f C C K - 8 suggests that a m i n o p e p t i d a s e A may cleave the peptide but this enzyme is undetectable in synaptic m e m b r a n e preparations. Surprisingly, we observed that affinity

purified A P - N , which has a marked preference for Nterminal amino acids with neutral side chains (Ala, Leu), hydrolysed C C K - 8 effectively. Since this enzyme is present in synaptic m e m b r a n e preparations and is responsible for enkephalin metabolism along with N E P (Turner et al., 1987), it may represent the previously detectcd aminopeptidasc cleaving C C K (Matsas et al., 1984). However, no entirely selective inhibitors of this enzyme are yet available to test this hypothesis directly (AM inhibits several aminopeptidases, including A and N). Consistent with these observations we have also seen that A P - N efficiently cleaves two substance P antagonists ( M E N 10376 and M E N 10207) which both possess an N-

Fig. 5. (opposite) HPLC analysis of CCK-8ns (50 pM) metabolism by D384 and SH-SY5Y cell membrane preparations. CCK-8ns (50/~M) was incubated with D384 and SH-SY5Y membrane preparations for 6 h at 37~'C. Products were resolved by HPLC as described in Experimental Procedures. Metabolism of CCK8ns is shown by: (a) SH-SY5Y cell membranes (114 ~g protein, hydrolysis = 80.6%); (b) SH-SY5Y cell membranes (114 #g protein, hydrolysis = 19.6%)+ 10 /~M MK 422: (c) SH-SY5Y cell membranes (114 #g protein, hydrolysis=76.4%)+l mM Dip-F; (d) D384 cell membranes (50 ~tg protein, hydrolysis = 14.7%).

Cholecystokinin metabolism in cell lines

375

CCK-Sns C.

CL. CCK-Sns

A 21b,

JL

L I

10

15

20

2O

0

CCK-8ns

b.

:CK-Ons

d.

1

A21a

~a k,...,-~

16

1'5

2O TIME ( min )

Fig. 5--legend opposite.

376

MARGARET DOS SANTOS MEDEIROS a n d ANTHONY J. TURNER Table 4. Effect of inhibitors on metabolism of CCK-8ns by solubilized preparations of SH-SY5Y cell membranes Product elution time (min) 5.54 6.58 8.32 10.50

Inhibition (%) -10 #M PR

10 #M MK 422

10 I,M AM

I mM Dip-F

43.7 25.6 39.9 20.1

74.6 86.2 88.6 84.4

38.5 2.9 0 7.7

12.1 0 0 0

CCK-Sns (50 #M) was incubated with 114 ,ug protein of SH-SY5Y cell membranes for 6h (hydrolysis - 80.6%) and in the presence of inhibitors. The products were analysed by reverse-phase HPLC as described in Experimental Procedures.

terminal A s p - T y r bond (M. S. Medeiros and A. J. Turner, unpublished observations). A C E is a third cell-surface peptidase capable of metabolising C C K in vitro. In the present studies it was noteworthy that the relative rates of hydrolysis of CCK-8 sulphated and non-sulphated by N E P did not differ substantially, consistent with the point of sulphation (Tyr:) being relatively distant from the primary cleavage site. The converse was true with A C E and A P - N which is not unexpected in the latter case as it is the Asp ~ Tyr 2 bond which is cleaved. With A C E , the sulphation of Tyr: must affect binding or hydrolysis although this residue is distant from the initial cleavage site. This may represent an unfavourable interaction of the sulphate with a critical residue in or near the active site of ACE. Evidence implicating a serine endopeptidase in C C K metabolism has come from the ability of serine peptidase inhibitors to protect C C K from metabolism when released by depolarisation of rat brain slices (Rose et al., 1988, 1989: Camus et al., 1989). Direct evidence for this serine peptidase in membrane fractions or cells is lacking. The renal microvillar membrane has often served as a model system for characterization of cell-surface peptidases (Kenny et al., 1987). The majority of microvillar peptidases are metalloenzymes. In this membrane the only detectable serine peptidase is DPP-IV which does not hydrolyse CCK. A tryptic-like serine endopeptidase (hepsin) has been reported in liver plasma membranes but this does not have the correct specificity for CCK hydrolysis (Torres-Rosado et al., 1993). Thus the nature of the putative serine endopeptidase in brain is unclear. In order to provide additional supporting evidence for such a putative serine endopeptidase we have made use of two human cell lines, one of neuronal and one of glial origin and have examined C C K metabolism by solubilized membrane preparations from these cell lines. We have consistently failed to detect significant inhibition of C C K metabolism by the general serine peptidase inhibitor Dip-F in either cell line. In contrast, in the glial cell line, the N E P inhibitor PR

completely abolished C C K metabolism. In the neuroblastoma line a major contribution due to A C E was seen with a lesser contribution from NEP. Little effect was attributable to aminopeptidase activity. The levels of N E P in the neuroblastoma line examined here and in other neuronal lines vary from low to undetectable, depending on culture conditions and passage number (Medeiros et al., 1991). In the present studies the levels of N E P in the neuroblastoma line were less than 2% ot" those in the glial cell line and hence this might have facilitated detection of previously unobserved peptidase activities. Nevertheless, only A C E was seen as an additional contributor to CCK degradation in this cell line. The ability of peptidase inhibitors to increase the levels of C C K in the superfusate of brain slices may be due to inhibition of degradation in the extracellular medium. However, caution is needed in the interpretation of such experiments since other parameters, e.g. peptide release, may be affected. The failure of Dip-F to affect C C K metabolism in either cell line argues against a predominant role for such a serine proteinase in CCK metabolism in these systems. At least in some brain regions it remains likely that N E P and/or A C E are important inactivating enzymes for this neuropeptide. Both these enzymes are widely distributed and are present in the gastro-intestinal tract and pancreas and may therefore also contribute to C C K inactivation in these peripheral tissues. Acknowled.qemems We thank the M.R.C. for financial support, Dr P. F. T. Vaughan and Dr A. J. Balmforth tk)r the generous supply of cell lines. We arc grateful to Mrs L. Backhouse for her expert secretarial assistance. REFERENCES

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