Life Sciences, Vol. 50, pp. 667-675 Printed in the USA

ENKEPHALIN Shinji

Pergamon Press

HYDROLYSIS

BY MOUSE PLASMA IN VITRO

Shibanoki 1, Susan B. Weinberger 2, Gery Schulteis 2, Koichi Ishikawa I, and Joe L. Martinez, Jr. 2

iDepartment of Pharmacology, Nihon University School of Medicine, Tokyo, Japan; 2Department of Psychology, university of California, Berkeley, CA (Received in final form January 3, 1992) Summary Hydrolysis of [Leu]- and [Met]enkephalin was determined in samples of pooled whole mouse plasma in vitro by using HPLC-ECD to measure accumulation of Tyrcontaining metabolites. More Tyr-Gly-Gly accumulated from [Met]enkephalin than from [Leu]enkephalin hydrolysis, and [Met]enkephalin's half-life in mouse plasma was approximately half that of [Leu]enkephalin. Comparisons of metabolite formation in the presence versus the absence of inhibitors with high selectivity for various peptidases demonstrated that a bestatinsensitive aminopeptidase, presumably aminopeptidase M, as well as enkephalinase and angiotensin converting enzyme, participate in the hydrolysis of enkephalin in mouse plasma. Peripheral administration of enkephalin and other opioid peptides leads to powerful effects on learning and memory in several mammalian species (see 1 and 2 for recent reviews). Recent studies in our laboratory suggest that understanding the enzymatic hydrolysis of enkephalin in plasma may be important to understanding some of its effects on conditioned behavior. For example, the overall rate of [Leu]enkephalin hydrolysis by rat plasma in vitro changes in association with training on a one-way active avoidance task. Both increases and decreases in hydrolysis rate were noted when blood samples collected from individual rats before and after the first avoidance training trial were compared (3). Studies in mice indicate that administration of [Leu]enkephalin metabolites containing at least an N-terminal Tyr-Gly sequence results in an impairment of one-way active avoidance conditioning similar to that produced by [Leu]enkephalin itself (4-6). In order to interpret the conditioning effects produced in mice by enkephalin and its Tyr-containing metabolites, it is necessary to determine which enyzmes normally participate in enkephalin hydrolysis in mouse plasma. Previous studies indicate

0024-3205/92 $5.00 + .00 Copyright © 1992 Pergamon Press plc All rights reserved.

668

Enkephalin Hydrolysis by Mouse Plasma

Vol. 50, No. 10, 1992

that [Leu]enkephalin has a short (2-2.5 min) half-life in rat plasma in vitro (7,8) and an even shorter (less than 1 min) halflife in vivo when administered i.p. to rats (9). A m i n o p e p t i d a s e M and a n g i o t e n s i n converting enzyme appear to be the major peptidases that hydrolyze enkephalins in rat plasma (7,8). By contrast, [Leu]enkephalin is hydrolyzed more rapidly by chick plasma than by rat plasma in vitro (half-life 0.7-1 min in chick plasma), and a b e s t a t i n - s e n s i t i v e aminopeptidase, p r e s u m a b l y a m i n o p e p t i d a s e M, accounts for over 99% of the in vitro enkephalin hydrolysis by plasma collected from the chick (i0). In the present study we used high performance liquid c h r o m a t o g r a p h y with electrochemical detection (HPLC-ECD) to characterize the time course of, and the enzymes involved in, the hydrolysis of [Leu]- and [Met]enkephalin by mouse plasma i_nn vitro. Previous studies in our laboratory indicated that [Leu]enkephalin is rapidly taken up and hydrolyzed in vivo following its i.p. or s.c. administration to mice (Janak, Schulteis, and Martinez, submitted for publication). As detailed below, the results of the present study indicate that, as in rats and chicks, a b e s t a t i n - s e n s i t i v e aminopeptidase, p r e s u m a b l y aminopeptidase M, is the primary enzyme that hydrolyzes enkephalin in mouse plasma in vitro. However, hydrolysis is considerably slower in the mouse than it is in the rat or the chick, and dipeptidyl carboxypeptidases play a larger role in enkephalin hydrolysis by mouse plasma than they do in the hydrolysis of enkephalin by rat or chick plasma. Methods Animals. The subjects were male Swiss-Webster mice (Harlan Sprague-Dawley, Inc., Indianapolis, IN) aged 56-60 days on arrival. The animals were housed in groups of three under standard laboratory conditions, in accordance with NIH guidelines. All experimental procedures were approved in advance by the Institutional Animal Care and Use Committee at the U n i v e r s i t y of California at Berkeley. Experiments were completed within one week of the animal's arrival at our colony. Blood Collection. Animals were brought to the testing room at least one hour prior to sample collection. They were sacrificed by decapitation and trunk blood was collected into h e p a r i n i z e d m i c r o c e n t r i f u g e tubes. Plasma was separated by m i c r o c e n t r i f u g a t i o n and incubated at 37°C for at least I0 mins to permit hydrolysis of endogenous enkephalins. Aliquots of plasma from 5-6 mice were pooled for each assay. Plasma Hydrolysis of [Leu]- and rMet]enkephalin. Aliquots (115 ~i) of pooled plasma were combined with saline (i0 ~i) containing one or more of the following peptidase inhibitors: bestatin (prepared in saline to achieve a final concentration of 125-500 BM), puromycin (prepared in saline to achieve a final c o n c e n t r a t i o n of 500 or 5000 ~M), thiorphan (prepared in 5% N,Ndimethyl formamide in saline in achieve a final concentration of

Vol. 50, No. 10, 1992

Enkephalin Hydrolysis by Mouse Plasma

669

i00 ~M), and captopril (prepared in saline to achieve a final concentration of i00 ~M). Following addition of the appropriate inhibitors, the samples were incubated for i0 min at 37°C. Either [Leu]- or [Met]enkephalin (prepared in saline to achieve a final concentration of 64.8 ~M) then was added to the plasma samples, followed by an additional 2-20 min incubation period. The reaction was stopped by the addition of two volumes of trichloroacetic acid (5% v/v) containing an appropriate internal standard ([Leu]- or [Met]enkephalin, or Tyr-Ala, for substrate and metabolite assays, respectively). Following microcentrifugation, 50 ~i of the supernatant were injected into the HPLC system for analysis. Chromatoqraphy Conditions. Procedures for HPLC-ECD m e a s u r e m e n t of enkephalins and metabolites are detailed in Shibanoki et al. (ii). Briefly, the system is comprised of an ISCO (Lincoln, NE) Model 2350 ternary pump, an ISCO Spherisorb C18 column (4.6 x 250 mm), and an Eicom (Kyoto, Japan) carbon graphite electrochemical detector. The column was maintained at 25°C throughout the separation. For assay of [Leu]- and [Met]enkephalin, the mobile phase was comprised of 0.05 M sodium citrate~citric acid buffer (pH 6.3) containing 19% acetonitrile, the flow rate was 1.2 ml/min, and the applied voltage was set at 1050 mV versus the Ag/AgCl reference electrode. For assay of Tyr, Tyr-Gly, and Tyr-Gly-Gly, a 0.05 M sodium citrate/citric acid buffer (pH 6.3) mobile phase was used, the flow rate was 1.4 ml/min, and the applied voltage was set at 950 mY. Chemicals. Bestatin, puromycin, Tyr, Tyr-Gly, Tyr-Gly-Gly, and Tyr-Ala were purchased from Sigma (St. Louis, MO); thiorphan was purchased from Peninsula Labs (Belmont, CA); [Leu]- and [Met]enkephalin were purchased from Bachem (Torrance, CA); and captopril was donated by the Squibb Institute for Medical Research (Princeton, NJ). Data Analysis. Endogenous [Leu]- and [Met]enkephalin, Tyr-Gly, and Tyr-Gly-Gly levels were not detectable with this assay system (detection limit 0.03 nmol/ml plasma), while endogenous Tyr concentrations were found to be 75-100 nmol/ml of mouse plasma. The amount of Tyr formed as a result of hydrolysis of exogenously added enkephalins was estimated by calculating the difference in the Tyr concentration in the presence versus the absence of added enkephalin. Preplanned comparisons involving dependent t-tests were used to evaluate the significance of the differences in substrate disappearance and in metabolite accumulation in plasma samples incubated with various combinations of substrates and inhibitors. As discussed in Keppel (12), when the number of comparisons is less than the degrees of freedom, Type I error is not inordinately increased.

670

Enkephalin Hydrolysis by Mouse Plasma

Vol. 50, No. 10, 1992

Results H y d r o l y s i s of [Leu]- and [Met]enkephalin. As m e a s u r e d by the amount of intact substrate r e m a i n i n g between 2 and 20 min after e n k e p h a l i n addition, the h a l f - l i v e s of [Leu]- and [Met]enkephalin in mouse p l a s m a in vitro were estimated to be 9.1 and 4.6 min, respectively. A s s a y of the amounts of each T y r - c o n t a i n i n g m e t a b o l i t e a c c u m u l a t e d at these same time points indicated that similar enzymes h y d r o l y z e [Leu]- and [Met]enkephalin in mouse plasma, but that there are d i f f e r e n c e s in the relative c o n t r i b u t i o n s of these enzymes to the overall h y d r o l y s i s of the two e n k e p h a l i n s (see FIG. i). Free Tyr was the p r e d o m i n a n t m e t a b o l i t e d e t e c t e d following h y d r o l y s i s of both enkephalins; after a p p r o x i m a t e l y one h a l f - l i f e it accounted for 78% of the T y r - c o n t a i n i n g [Leu]enkephalin m e t a b o l i t e s and 67% of the T y r - c o n t a i n i n g [Met]enkephalin metabolites. Only very small amounts (1-1.2% of the total T y r - c o n t a i n i n g metabolites) of T y r - G l y a c c u m u l a t e d from the h y d r o l y s i s of both enkephalins. By contrast, 20% of the T y r - c o n t a i n i n g [Leu]enkephalin m e t a b o l i t e s and 32% of the T y r - c o n t a i n i n g [Met]enkephalin m e t a b o l i t e s were in the form of T y r - G l y - G l y by the end of a p p r o x i m a t e l y one halflife. These results indicate a larger role for a m i n o p e p t i d a s e a c t i v i t y in [Leu]enkephalin than in [Met]enkephalin hydrolysis,

LE 60

~o

0

10

20

ME

o

_E so

~"

2040 i~ ~o" 0 m

•

Incubation

10

20

/

time

I

(min)

FIG. 1 Time course of the d i s a p p e a r a n c e of substrate ([Leu]enkephalin [LE] or [Met]enkephalin [ME], 64.8 ~M initial concentration, O O) and a c c u m u l a t i o n of p r o d u c t (Tyr, o - - o ; Tyr-Gly, B - - B ; and Tyr-Gly-Gly, A--A) in mouse plasma in vitro. Values are means for four p o o l e d plasma samples.

Vol. 50, No. I0, 1992

Enkephalin Hydrolysis by M o u s e P l a s m a

671

a n d a l a r g e r r o l e for d i p e p t i d y l c a r b o x y p e p t i d a s e a c t i v i t y in [ M e t ] e n k e p h a l i n t h a n in [ L e u ] e n k e p h a l i n h y d r o l y s i s , by m o u s e p l a s m a in vitro. S t u d i e s w i t h p e p t i d a s e - s e l e c t i v e inhibitors were u n d e r t a k e n t o a t t e m p t to i d e n t i f y t h e s p e c i f i c e n z y m e s i n v o l v e d in t h i s h y d r o l y s i s . R o l e of A m i n o p e p t i d a s e A c t i v i t y in t h e H y d r o l y s i s of [Leu]and [Met]enkephalin. In the p r e s e n c e of t h e a m i n o p e p t i d a s e i n h i b i t o r , b e s t a t i n (13,14), a s i g n i f i c a n t c o n c e n t r a t i o n d e p e n d e n t d e c r e a s e in T y r a c c u m u l a t i o n f r o m [Leu]- a n d [ M e t ] e n k e p h a l i n w a s o b s e r v e d (see T a b l e i). A 9 8 - 9 9 % r e d u c t i o n in T y r a c c u m u l a t i o n f r o m b o t h e n k e p h a l i n s w a s p r o d u c e d by t h e 500 ~ M c o n c e n t r a t i o n of b e s t a t i n . T h i s r e d u c t i o n in T y r f o r m a t i o n w a s a c c o m p a n i e d by a s i g n i f i c a n t c o n c e n t r a t i o n - d e p e n d e n t i n c r e a s e in b o t h T y r - G l y a n d T y r - G l y - G l y a c c u m u l a t i o n . A g r e a t e r i n c r e a s e in T y r - G l y - G l y a c c u m u l a t i o n w a s o b s e r v e d in t h e p r e s e n c e of b e s t a t i n f o l l o w i n g a d d i t i o n of [ M e t ] e n k e p h a l i n , t h a n f o l l o w i n g a d d i t i o n of [Leu]enkephalin, to m o u s e plasma. P u r o m y c i n , a s e l e c t i v e i n h i b i t o r of a m i n o p e p t i d a s e MII (13,14), p r o d u c e d a s i g n i f i c a n t r e d u c t i o n in T y r a c c u m u l a t i o n f r o m [Leu]- a n d [ M e t ] e n k e p h a l i n at a final c o n c e n t r a t i o n of 5000, b u t n o t of 500, ~M. T h e h i g h e r p u r o m y c i n c o n c e n t r a t i o n p r o d u c e d a 4 8 - 6 3 % d e c r e a s e in f o r m a t i o n of free Tyr. In c o n t r a s t to t h e T A B L E i. E f f e c t of B e s t a t i n Tyr-Gly, and Tyr-Gly-Gly Substrate

Inhibitor Tyr

LE a

None

Product (nmoles/ml Tyr-Gly + 1.6

0.79

+ 0.14

plasma) Tyr-Gly-Gly 4.39

+ 0.32

125 ~ M b B 250 ~M B 500 ~M

4.0 + 1 . 9 . 1.4 _+ 0.9* 0.2 _+ 0.2*

1.24 _+ 0.18, 1.37 _+ 0.35* 1.48 + 0.25*

8.83 _+ 1.099.59 _+ 1.19, 9.89 + 0.97*

P 500 ~M c P 5000 ~ M

29.1 _+ 3.2 15.6 _+ 1.8"

0.87 + 0.05 0.44 _+ 0.17,

5.71 + 0.88 11.51 _+ 0.90*

None

45.3

0.79

B

ME d

32.7

a n d P u r o m y c i n on F o r m a t i o n of Tyr, f r o m [Leu]- a n d [ M e t ] e n k e p h a l i n

B 125 ~ M B 250 ~ M B 500 ~ M P 500 ~ M P 5000 ~ M

+ 1.5

+ 0.16

8.61

+ 1.36

5.1 + 1.3" 1.4 _+ 1.6" 1.0 + 0.9*

1.30 _+ 0.30* 1.53 + 0.27* 1.55 + 0.16,

19.81 20.18 21.63

_+ 2.07* _+ 1.23, _+ 1.70-

41.9 _+ 2.8 28.4 _+ 3.3*

0.87 _+ 0.15 0.48 _+ 0.15,

10.90 18.30

_+ 2.71 + 1.04,

a[Leu]enkephalin; b B e s t a t i n ; Cpuromycin; d [ M e t ] e n k e p h a i i n Substrate concentration: 64.8 ~M; I n c u b a t i o n time: I0 min. V a l u e s a r e m e a n s ± S.D. for f o u r p o o l e d p l a s m a samples. * p < 0 . 0 5 c o m p a r e d to "none" group.

672

Enkephalin Hydrolysis by Mouse Plasma

Vol. 50, No. i0, 1992

pattern of metabolite accumulation observed in the presence of bestatin, a reduction of Tyr accumulation from hydrolysis of both enkephalins in the presence of puromycin was accompanied by a significant increase in Tyr-Gly-Gly accumulation and a significant reduction in Tyr-Gly accumulation. A combination of bestatin (500 BM) and puromycin (5000 ~M) resulted in a reduction in Tyr accumulation from both enkephalins similar to that produced by bestatin alone, but significantly greater than that produced by puromycin alone. Role of Dipeptidyl Carboxypeptidase Activity in the Hydrolysis of [Leu]- and [Met]enkephalin. The contribution of dipeptidyl carboxypeptidase activity to the hydrolysis of [Leu]and [Met]enkephalin was studied in the presence of the aminopeptidase inhibitor, bestatin (500 ~M), in order to minimize aminopeptidase activity against the Tyr-Gly and Tyr-Gly-Gly metabolites. Thiorphan (i00 ~M), an inhibitor of "enkephalinase" (E.C. 3.4.24.11) (15,16), significantly and markedly reduced Tyr-Gly-Gly formation from both enkephalins in the presence of

~

LE

ME

1.0

m Q.

0

TGG

c

".:

.:

0

o ; o +

o ; o

+

+

+ +

Inhibitor

FIG.

2

Effects of captopril (C; i00 #M) and thiorphan (T; i00 ~M) on the accumulation of Tyr-Gly (TG) and Tyr-Gly-Gly (TGG) in mouse plasma, measured i0 min after addition of [Leu]- or [Met]enkephalin (LE or ME, 64.8 ~M) in the presence of bestatin (500 ~M). Values are means ± S.D. for six pooled plasma samples. ~ p