49

Biochimica et Biophysica Acta, 500 (1977) 49--60 Q Elsevier/North-Holland Biomedical Press

BBA 28323 E F F E C T OF LOCAL ANESTHETICS ON PLASMA PROTEIN SECRETION BY RAT HEPATOCYTES

D. BANERJEE and C.M. REDMAN

The Lindsley F. Kimball Research Institute o f The New Yo'rk Blood Center, 310 East 67th Street, New York, N.Y. 10021 (U.S.A.)

(Received March 4th, 1977)

Summary The effects of some local anesthetics on plasma protein secretion by rat liver slices have been studied and have been compared with those of colchicine. Rat liver slices were pulse-labelled with L- ['4C ]leucine for 9 min at 37 ° C, collected on filter paper, washed with non-radioactive leucine and reincubated in the presence or absence of the drug to be tested. The radioactive plasma proteins produced were obtained by immunoprecipitation from either the chase medium or from the washed slices. Chlorpromazine, ( 3 . 1 0 -s M), dibucaine (10 -s M), lidocaine (10 -3 M) and procaine (5 • 10 -s M) inhibited both the synthesis and secretion of plasma protein but did not affect the uptake of L-leucine into the slices nor the incorporation of phosphate into intracellular nucleotide phosphates or into phospholipids. The inhibition of secretion elicited by these drugs is probably n o t due to the inhibition of protein synthesis since cycloheximide, when added to the chase medium at a concentration which completely inhibits protein synthesis, did n o t inhibit plasma protein secretion, while cycloheximide plus procaine did inhibit secretion and also caused a retention of non-secreted plasma proteins within the slices. Unlike colchicine, however, procaine did n o t cause the retained plasma proteins to accumulate in Golgi-derived secretory vesicles, but showed a more general effect causing a distribution among several cell fractions.

Introduction Our previous studies have shown t h a t colchicine inhibits plasma protein secretion both in vivo and in liver slices, and causes the hepatocytes to accumulate non-secreted plasma proteins within Golgi-derived secretory vesicles without affecting other steps in the secretory process [1]. Other studies [2] have also demonstrated that colchicine inhibits secretion in a variety of other tissues and most of these reports have implicated the participation of cytoplasmic

50 microtubules in the secretory phenomenon based on the fact that colchicine is known to bind to tubulin and to interfere with in vitro polymerization of tubulin into microtubules. The exact mechanisms by which colchicine affects secretion in these tissues is still not known, however. Besides the well known antimicrotubular agents such as vinblastine and colchicine, the phenothiazine tranquillizer, chlorpromazine, and several local anesthetics such as procaine, lidocaine and xylocaine, have also been shown to affect cellular microtubules and microfilaments [3]. It has been further reported that lidocaine like colchicine inhibits in vitro microtubule polymerization [4]. Local anesthetics do not, however, have the antimicrotubular specificity usually ascribed to colchicine and they have also been shown to produce a variety of effects on membranes; they interact with lipids [5--12], displace calcium [6,13,14] and expand membranes [14], inhibit cell specific movement [15,16,17] and affect the adhesion [18,19], and fusion of cells [20,21,22]. Since colchicine and the local anesthetics share the ability to disrupt cytoplasmic microtubules, we have compared their effects on plasma protein secretion by rat liver slices to determine whether the local anesthetics inhibit secretion and if, like colchicine, the inhibition of secretion is limited to affecting the discharge of plasma protein from Golgi-derived secretory vesicles. Materials and Methods

Preparation and incubation o f liver slices Slices, approximately 0.5 mm in thickness, were prepared with a StadieRiggs hand slicer. About 0.3 g of slices were pulse-labelled by incubating at 37°C for 9 min in 5 ml of Krebs-Ringer bicarbonate saline supplemented with 0.21 M glucose and a mixture of 20 amino acids (0.1 mM) minus leucine, which was provided as L-[U-14C]leucine, (0.25 pCi/ml, specific activity 348 mCi/ mmol). The slices were then recovered by filtration, washed with 0.7 mM nonradioactive L-leucine in 0.154 M NaC1 and reincubated at 37°C in 5 ml KrebsRinger bicarbonate containing 0.21 M glucose and a 0.1 mM mixture of 20 non-radioactive amino acids which included non-radioactive L-leucine. The slices were then removed by centrifugation and the secreted proteins were recovered from the incubation medium either by precipitating with trichloroacetic acid or by immunoprecipitation. The proteins retained within the tissue were recovered by similar means after the slices were washed free of incubation medium by three successive changes with 0.154 M NaC1 [1]. Cell fractionation procedures Liver slices (4--6 g) were homogenized in 5 volumes of ice cold 0.25 M sucrose with 5--6 strokes of a Potter-Elvejehm homogenizer with a teflon pestle, m o t o r driven at 1000 rev./min. The homogenate was filtered through two layers of cheesecloth and centrifuged at 166500 × g for 1 h. The resulting supernatant was used as a "soluble" cell fraction. This first centrifugation, in which all of the membrane fractions are pelleted, was found to be necessary for cell fractionation of liver slices which are incubated in Kreb-Ringer bicarbonate. If this procedure is n o t followed, the nuclear, mitochondrial and microsomal cell fractions tend to sediment at lower centrifugal forces than are nor-

51 mally used for cell fractionation o f rat liver. The 166 500 X g m em brane pellet thus obtained was then resuspended in 0.25 M sucrose to the original homogenate volume with the aid of a Potter-Elvejhem hom ogeni zer and a " n u c l e a r " fraction was isolated by centrifugation at 800 X g for 10 min. F r o m the resulting supernatant, a " m i t o c h o n d r i a l " cell fraction was obtained by centrifugation at 12 100 × g for 10 min. A total " m i c r o s o m a l " fraction, i.e., a m i xt ure of microsomes and Golgi elements, was obtained by centrifugation at 166 500 X g for 90 min. This latter microsomal cell fraction was then furt her fractionated into a rough endoplasmic reticulum fraction, a s m o o t h endoplasmic reticulum fraction and into three Golgi fractions as previously described [1]. The a m o u n t of radioactive plasma protein in each of the cell fractions was d e t er min ed by i m m u n o p r e c i p i t a t i o n . The cell fractions were treated as follows prior to i m m u n o p r e c i p i t a t i o n . The " n u c l e a r " , " m i t o c h o n d r i a l " and " m i c r o s o m a l " pellets were resuspended in 0.154 M NaC1 buffered with 0.01 M T r i s . HC1, pH 7.4, to which sodium d e o x y c h o l a t e was added to a final c o n c e n t r a t i o n of 0.5%. The samples were then cleared of non-solubilized material by centrifugation at 1 6 6 5 0 0 X g for 1 h. When determining the am ount s of radioactive plasma proteins in the h o m o g e n a t e , a sample corresponding to 0.1 g liver was diluted with 0.154 M NaC1 (buffered with 0.01 M Tris • HC1, pH 7.4) to 10 ml and was then treated with 0.5% sodium d e o x y c h o l a t e and cleared of insoluble material by centrifugation at 1 6 6 5 0 0 X g for 1 h. The " s o l u b l e " cytoplasmic fraction was n o t treated with sodium d e o x y c h o l a t e b u t was treated with Tris and NaC1 so t hat the final concentrations were 0.01 M T r i s . HC1, pH 7.4, and 0.154 M NaC1. The " c h a s e " med i um in which slices had been incubated was used w i t h o u t f u rt h er t r e a t m e n t .

Immunoprecipitation assay Radioactivity in plasma proteins or in albumin was determined in the above cell fractions by i m m u n o p r e c i p i t a t i o n using either a multivalent rabbit antiserum to rat plasma proteins or a m o n o v a l e n t rabbit antiserum to purified rat serum albumin. This was preceded by a "clearing" t r e a t m e n t of the samples with chicken serum and rabbit antiserum to chicken serum in order to reduce the non-specific i m m u n o p r e c i p i t a t i o n of radioactive material. The preparation of the antisera and t he m e t h o d s used t o i m m u n o p r e c i p i t a t e these proteins have been previously described [ 1].

Other procedures Radioactive samples, w het her t he y were trichloroacetic acid-precipitated proteins or immunoprecipitates, were dissolved in 1 ml o f Soluene ® and c o u n t e d in a Packard scintillation r adi om e t er in the presence of 10 ml of toluene scintillation p h o s p h o r and 0.1 ml of glacial acetic acid. Protein was assayed by the m e t h o d of L o w r y et al. [23], with bovine serum albumin as a standard. The specific activity of the intracellular nucleotide phosphates was determined by adsorption o f the nucleotides, obtained from a 5% trichloroacetic acid soluble fraction o f the h o m o g e n a t e , on charcoal and hydrolysis of the adsorbed material in 1 N HC1 for 10 min at 100°C as described by Crane and

52

Lipmann [24]. Phosphorus analysis was performed by the method of Bartlett [251.

Sources of material L-[U-I4C]Leucine (348 mCi/mmol) was purchased from Amersham/Searle Corp., Arlington Heights, Ill.; procaine hydrochloride and cycloheximide from Sigma Chemical Co., St. Louis, Mo.; dibucaine hydrochloride and lidocaine were obtained from Pfaltz and Baner, Inc., Flushing, N.Y. and chlorpromazine hydrochloride from Smith, Kline and French Laboratories, Philadelphia, Pa. Results

Effect of chlorpromazine, dibucaine, lidocaine and procaine on the synthesis and secretion of plasma proteins by liver slices Liver slices were pulse labelled with L-[14C]leucine for 9 min, washed and then reincubated for 90 min in a "chase" medium containing non-radioactive leucine and the anesthetic to be tested. The slices were removed by centrifugation and the radioactive proteins secreted into the medium were obtained by precipitation with trichloroacetic acid. Chlorpromazine (3 • 10 -~ M), dibucaine (10 -S M), lidocaine (4 • 10-: M) and procaine (5 • 1 0 - 4 M) inhibited the secretion of plasma proteins by 12--23%. These were the lowest drug concentrations found to cause inhibition of secretion, but greater inhibitions (63--88%) were obtained at higher anesthetic concentrations (Table I). To test whether concentrations of these drugs which markedly affected TABLE EFFECT

I OF LOCAL

ANESTHETICS

ON PLASMA

PROTEIN

SECRETION

BY RAT LIVER

SLICES

R a t liver s l i c e s w e r e p u l s e 4 a b e l e d with L-[14C]leucine ( 0 . 2 5 p C i / m l ) f o r 9 rain a n d t h e n " c h a s e d " w i t h non-radioactive Krebs-Ringer bicarbonate medium containing the above concentration o f d r u g s for 9 0 rain. The L-[ 14C]leucine-labeled serum proteins secreted into the "chase" medium were determined by t r i c h l o r o a c e t i c a c i d p r e c i p i t a t i o n . T h e d e t a i l s o f t h e s e p r o c e d u r e s are g i v e n in M a t e r i a l s a n d M e t h o d s . Plasma protein secretion (cpm/mg slice protein)

Inhibition

Expt. 1

Control Chlorpromazine

400 3

10 -5 M

309

23

3

10

150

63

1

1 0 -5 M

4M

Expt. 2

Control Dibucaine

176 142 43

19 76

1.25 • 10 -3 M 4 - 10 -2 M

374 295 116

21 69

5 • 10 -4 M 5 • 10 -3 M 5 • 10 -2 M

779 683 223 90

1 - 10-4

M

Expt. 3

Control Lidocaine Expt. 4

Control Procaine

12 71

88

(%)

53 secretions also inhibited protein synthesis, their effect on the incorporation of L-['4C]leucine into trichloroacetic acid-precipitable proteins of liver slices, was measured. Slices were incubated for 1 h in the presence or absence of the anesthetic. Incorporation of radioactivity into total trichloroacetic acid-precipitable protein, both within the slice and secreted into the medium, was linear during this time. All of the anesthetics tested inhibited protein synthesis to varying extents with lidocaine (3 • 10 -4 M) virtually completely inhibiting synthesis and procaine (5 • 10 -3 M) having the least (26%) inhibitory effect (Table II).

Lack o f effect o f anesthetics on the uptake o f L-['4C] leucine by liver slices The assay used to determine protein synthesis and secretion is based on the incorporation of L-['4C]leucine into proteins and this can be affected if the anesthetics hamper the uptake of this amino acid by the liver slices. Thus slices were incubated with L-['4C]leucine in the presence and absence of anesthetics and the slices were removed at various time intervals, quickly washed with saline to remove the radioactive incubation medium, and the L-[14C]leucine taken up b y the slices was measured by determining the radioactivity in a 5% trichloroacetic acid-soluble fraction of homogenized slices. A representative experiment, using chlorpromazine, is shown in Fig. 1, and in experiments using dibucaine (10 -4 M), and procaine (5 • 10 -3 M) although n o t shown in the figure, gave similar results. L-['4C]leucine was quickly taken up by the liver slices, reaching a maximal level by 15 min and the intracellular trichloroacetic acidsoluble radioactivity remained at this level for 1 h, which was the duration of the experiments. The drugs tested did n o t affect the uptake of L-['4C]leucine into the slices.

T A B L E II E F F E C T O F L O C A L A N E S T H E T I C S O N P R O T E I N S Y N T H E S I S BY R A T L I V E R S L I C E S R a t liver slices w e r e l a b e l e d c o n t i n u o u s l y w i t h L - [ 1 4 C ] l e u c i n e f o r 6 0 rain as d e s c r i b e d in M a t e r i a l s a n d M e t h o d s . D r u g s w e r e a d d e d a t z e r o t i m e . A t t h e e n d o f i n c u b a t i o n , t h e slices a n d t h e i n c u b a t i o n m e d i u m w e r e h o m o g e n i z e d a n d t r i e h l o r o a c e t i c a c i d - p r e c i p i t a b l e r a d i o a c t i v i t y w a s d e t e r m i n e d as d e s c r i b e d in Materials and Methods. Protein radioactivity ( c p m / m g o f slice p r o t e i n )

I n h i b i t i o n (%)

Expt. 1 Control Chlorpromazine

3.10 ~ M

1928 395

80

Expt. 2 Control Dibucaine

1 " 1 0 -4 M

1212 196

84

Expt. 3 Control Lidocaine

4.10

-2 M

805 10

99

Expt. 4 Control Procaine

5.10

-3 M

3737 2755

26

54

_Z 6 w

~4 ? o E2 Q.

I

10

I

20

30 MINUTES

I

40

50

60

F i g . 1. E f f e c t o f c h l o r p r o m a z i n e o n L - [ 1 4 C ] l e u c i n e u p t a k e i n t o r a t l i v e r s l i c e s . R a t l i v e r s l i c e s w e r e i n c u b a t e d w i t h L-[ 1 4 C ] l e u c i n e as d e s c r i b e d i n M a t e r i a l s a n d M e t h o d s . C h l o r p r o m a z i n e (3 • 1 0 -4 M) was a d d e d a t z e r o t i m e a n d t h e i n c u b a t i o n t i m e w a s v a r i e d f o r 6 0 m i n . A t t h e e n d o f i n c u b a t i o n t h e slices were w a s h e d 3 t i m e s w i t h ice c o l d s a l i n e . T h e w a s h e d s l i c e s w e r e h o m o g e n i z e d i n w a t e r a n d a l i q u o t s t a k e n f o r protein analysis and for determination of total trichloroacetic acid-soluble radioactivity, o--• Tric h l o r o a c e t i c a c i d - s o l u b l e r a d i o a c t i v i t y in c o n t r o l slices, a n d • . . . . . . •, trichloroacetic acid-soluble radioa c t i v i t y i n c h l o r p r o m a z i n e - t r e a t e d slices.

Effect of cycloheximide on plasma protein synthesis and secretion In testing the effect of the local anesthetics on plasma protein secretion, the rat liver slices were pulse-labelled with L-[14C]leucine, in the absence of drugs, and the drugs were i nt r oduc e d later to the " c h a s e " medium. Since in the " c h a s e " medium L-[14C]leucine has been removed and replaced with non-radioactive L-leucine, there should be little or no incorporation of radioactivity into proteins during this latter period when the drugs are present. Thus, the inhibit o r y ef f ect of the anesthetics on protein synthesis should not directly affect the incorporation of radioactivity into plasma proteins. However, since these drugs do inhibit protein synthesis and since inhibition of protein synthesis, even though it occurs after the nascent secretory proteins have been pulse-labelled, may affect secretion, we tested the e f f e c t of a known protein synthesis inhibitot, cycloheximide, on secretion. Sets of slices were incubated for 1 h in media containing L-[14C]leucine and concentrations of cycloheximide of 10 .6 M-1 0 - 3 M and the a m o u n t of radioactivity incorporated into the trichloroacetic acid-precipitable protein was measured. Cycloheximide inhibited protein synthesis by 80% at 10 .6 M, and by 96--98% at 10 -S and 10 .3 M. When sets of slices were pulse labelled for 9 min with L-[~4C]leucine and the labelled proteins were " c h a s e d " with non-radioactive leucine either in the presence or absence of increasing concentrations (10 -6 M--10 -4 M) of cycloheximide, there was no change in the p e r c e n t of radioactive plasma proteins secreted into the medium. At higher concentrations ( 5 . 1 0 - 4 M and 10 .3 M) there was some inhibition o f secretion as measured by trichloroacetic acid-precipitable protein radioactivity (Fig. 2). These experiments show t hat at 10 .6 to 10-4M cycloheximide, the transport o f secretory protein remains unaffected, though at the latter dose protein synthesis has virtually stopped. Therefore, since inhibition of protein synthesis by cycloheximide does n o t affect secretion, it seems un-

55

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60 u

•

I

0

-

-....,,~,

I

I

10-~ 10-5 10-4 CYCLOHEXIMIDE IM)

I

I0-3

F i g . 2. E f f e c t o f v a r i o u s c o n c e n t r a t i o n s o f c y c l o h e x i m i d e o n s e r u m p r o t e i n s e c r e t i o n by r a t liver slices. R a t liver slices w e r e p u l s e - l a b e l e d f o r 9 m i n w i t h L - [ 1 4 C ] l e u c i n e f o l l o w e d by a " c h a s e " f o r 90 m i n . V a r i o u s c o n c e n t r a t i o n s o f c y c l o h e x i m i d e w e r e a d d e d in t h e m e d i u m a t t h e b e g i n n i n g of t h e chase p e r i o d . The proteins secreted into the " c h a s e " m e d i u m were d e t e r m i n e d by i m m u n o p r e c i p i t a t i o n and by trichlor o a c e t i c a c i d p r e c i p i t a t i o n as d e s c r i b e d in M a t e r i a l s a n d M e t h o d s . T h e v a l u e s g i v e n in t h e f i g u r e r e p r e s e n t the p e r c e n t of r a d i o a c t i v e p r o t e i n s s y n t h e s i z e d w h i c h w e r e s e c r e t e d i n t o t h e m e d i u m . • •, Plasma proteins obtained by immunoprecipitation;. ., trichloroacetic acid-precipitable proteins.

likely that the inhibition of protein synthesis by local anesthetics will do it either. Further evidence for this was provided when serum albumin secretion was measured in slices which had been pulse-labelled with radioactive L-leucine and were then "chased" with either cycloheximide or with a combination of procaine plus cycloheximide. The addition of procaine to the "chase" medium caused a reduction in the secretion of radioactive albumin from 37.5% to 24%, and a c o n c o m i t a n t increase in non-secreted albumin within the slices from 62.5 to 76% (Table III). This inhibition of secretion occurred even though both sets of slices contained sufficient cycloheximide in the "chase" medium to completely inhibit protein synthesis. Similar results are shown in Fig. 6 where cycloheximide, present in the chase medium at a concentration that severely inhibits protein synthesis, still allows procaine to inhibit secretion and to cause a retention of the non-secreted protein in the slice.

Lack o f effect of chlorpromazine and procaine on the uptake o f inorganic phosphate and its incorporation into the nucleotide-phosphates and phospholipids We have shown that local anesthetics probably do not affect plasma protein secretion by inhibiting protein synthesis or by inhibiting the uptake of L-[14C] leucine into the slices. However, secretion may be affected by drug interference with other cellular parameters such as the intracellular level of ATP. This possibility was examined by incubating liver slices with radioactive inorganic 32p and measuring its incorporation into acid-labile organic phosphates, which may be considered a measure of its incorporation into [32P]ATP. Two drugs, chlorpromazine and procaine were used in these experiments. At various times from

56 TABLE nI I N T R A C E L L U L A R D I S T R I B U T I O N O F N O N - S E C R E T E D S E R U M P R O T E I N S IN P R O C A I N E - A N D COLCHICINE-TREATED SLICES R a t liver s l i c e s (6 g) w e r e p u l s e - l a b e l e d w i t h L-[ 1 4 C ] l e u c i n e f o r 9 m i n a n d t h e n " c h a s e d " f o r 90 rain w i t h K r e b s - R i n g e r b i c a r b o n a t e m e d i u m c o n t a i n i n g n o n - r a d i o a c t i v e L - l e u c i n e , 2.5 • 10 -4 M c y c l o h e x i m i d e a n d either 5 .10-]M procaine or 5-10-SM colchicine. The slices were collected by centrifugation and washed 3 times with 0.25 M sucrose. The washed slices were homogenized a n d f r a c t i o n a t e d as d e s c r i b e d in M a t e r i a l s a n d M e t h o d s . R a d i o a c t i v e s e r u m p r o t e i n s w e r e o b t a i n e d f r o m t h e i s o l a t e d cell f r a c t i o n s b y immunoprecipitation. In E x p t . 1 a n a n t i s e r u m t o r a t s e r u m w a s u s e d a n d in E x p t . 2 a n a n t i s e r u m t o purified rat a l b u m i n w a s u s e d . T h e n u m b e r s w i t h i n p a r e n t h e s e s s h o w t h e p e r c e n t a g e o f t o t a l r a d i o a c t i v e serum protein or a l b u m i n p r o d u c e d which was either retained in the slices or secreted into the m e d i u m . In E x p t . 1, 64% o f t h e r a d i o a c t i v e s e r u m p r o t e i n s r e t a i n e d i n t h e s l i c e s w e r e r e c o v e r e d in t h e cell f r a c t i o n s . I n E x p t . 2, 4 9 . 8 % w e r e r e c o v e r e d i n t h e c o n t r o l s l i c e s a n d 5 3 . 5 % in t h e c o l c h i c i n e - t r e a t e d s l i c e s . R a t i o represents experimental over control values. Experiment Activity

Secreted into Medium R e t a i n e d in Slices Retained

1

Experiment 2

( c p m / g slice)

Control

Procaine

8294(37.5) 13829(62.5)

4902(24) 15406(76)

A c t i v i t y ( c p m / g slice) Ratio

Control

Colchicine

3774(57) 2813(43)

1629(27) 4400(73)

Ratio

in Cell F r a c t i o n s

8 0 0 × g pellet 12 1 0 0 X g pellet Rough endoplasmic ulum

retic-

2150 1630 1698

2581 2943 1520

1.20 1.80 0.89

229 471 265

255 678 318

1.11 1.43 1.20

re-

2281

1768

0.77

218

442

2.02

ticulum Golgi F r a c t i o n 3 Golgi F r a c t i o n s 1 & 2 105 0 0 0 X t,* s u p e r n a t a n t

482 203 434

358 110 600

0.74 0.54 1.38

60 18 141

251 102 312

4.18 5.66 2.21

Smooth

endoplasmic

5 to 60 min the entry of 32Pi into the liver slices, its incorporation into phospholipids and its conversion into nucleotide phosphates were determined. Chlorpromazine and procaine had n o effect at any of the times tested, either on the specific activity of 32Pi in the slice, on its conversion to nucleotide phosphates, or on its incorporation into phospholipids. Figs. 3--5 summarize the results with chlorpromazine. The results using procaine (5 • 10 -3 M) were similar and are n o t s h o w n in the figures.

Retention of secretory proteins within the slices in the presence of procaine In our previous studies we have shown that the drug colchichine, given either intravenously to the rat or added to liver slices, inhibits the secretion of plasma proteins and causes a c o n c o m i t a n t accumulation of these proteins within the liver. To determine if a similar accumulation of secretory protein occurs inside the cell when plasma protein secretion is inhibited by procaine, we pulse labelled liver slices for 9 min with L-[14C]leucine, and "chased" for 90 min in the presence of cycloheximide to stop further protein synthesis. The a m o u n t of radioactive albumin secreted into the medium or retained in the washed slices was determined and is expressed (Fig. 6) as a percent of the total albumin synthesized. In control slices, about 4670 of the radioactive plasma proteins was secreted into the medium while the other 54% was retained within the slices. Procaine inhibited secretion, starting at 1 0 - 3 M with a maximal

57

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5

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x

u

u

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210

310 MINUTES

41

5~

610

I 20 30 MINUTES

10

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Fig. 3. U p t a k e o f i n o r g a n i c 3 2 p i n t o r a t liver slices. Slices w e r e i n c u b a t e d w i t h [ 3 2 P i ] as d e s c r i b e d in M a t e r i a l s a n d M e t h o d s in t h e p r e s e n c e a n d a b s e n c e o f c h l o r p r o m a z i n e (3 • 10 -4 M) f o r v a r y i n g p e r i o d s of t i m e u p t o 6 0 m i n . A t t h e e n d o f i n c u b a t i o n t h e slices w e r e r e c o v e r e d b y c e n t r i f u g a t i o n a n d w a s h e d 3 t i m e s w i t h i c e - c o l d saline. T h e w a s h e d slices w e r e h o m o g e n i z e d in i c e - c o l d 5% t r i c h l o r o a c e t i c a c i d a n d c e n t r i f u g e d . T h e s p e c i f i c r a d i o a c t i v i t y o f t h e i n o r g a n i c p h o s p h o r o u s in t h e t r i c h l o r o a c e t i c a c i d s o l u b l e f r a c t i o n w a s d e t e r m i n e d as d e s c r i b e d in M a t e r i a l s a n d M e t h o d s . • •, Control; • ..... -m, e h l o r p r o mazine. F i g . 4. E f f e c t o f c h l o r p r o m a z i n e o n t h e i n c o r p o r a t i o n o f 3 2 p i i n t o p h o s p h o l i p i d s of r a t liver slices. R a t liver slices w e r e i n c u b a t e d w i t h [ 3 2 p i] a n d p r o c e s s e d as d e s c r i b e d in t h e l e g e n d to F i g . 3. T h e p e l l e t o b t a i n e d f r o m t h e t r i c h l o r o a c e t i c a c i d p r e c i p i t a t i o n w a s w a s h e d 3 t i m e s w i t h c o l d 5% t r i c h l o r o a c e t i c a c i d a n d e x t r a c t e d w i t h C H C 1 3 / m e t h a n o l / H C l ( 2 0 0 : 1 0 0 : 1) a t 4 ° C o v e r n i g h t . T h e lipid e x t r a c t w a s f u r t h e r w a s h e d w i t h 0.1 N HC1 [ 2 7 ] a n d t h e s p e c i f i c r a d i o a c t i v i t y of t h e p h o s p h o l i p i d s w h i c h w e r e in t h e C H C I 3 layer was then determined by assaying for phospholipid radioactivity and for phospholipid phosphorous. S i m i l a r r e s u l t s w e r e o b t a i n e d if p r o c a i n e ( 5 " 1 0 - 3 M ) w a s u s e d i n s t e a d of c h l o r P r o m a z i n e . • e, Control; • ..... -m, c h l o r p r o m a z i n e .

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F i g . 5. S p e c i f i c r a d i o a c t i v i t y o f i n t r a c e l l u l a r n u c l e o t i d e p h o s p h a t e s in r e s p o n s e t o c h l o r p r o m a z i n e . Slices w e r e l a b e l l e d a n d p r o c e s s e d as d e s c r i b e d in t h e l e g e n d t o F i g . 3. T h e s p e c i f i c r a d i o a c t i v i t i e s of t h e i n t r a ceHul~Lr n u c l e o t i d e p h o s p h a t e s a t d i f f e r e n t t i m e s w e r e d e t e r m i n e d f r o m t h e t r i c h l o r o a c e t i c a c i d s o l u b l e f r a c t i o n as d e s c r i b e d in M a t e r i a l s a n d M e t h o d s . • • , C o n t r o l slices; • . . . . . . m chlorpromazinet r e a t e d slices. F i g . 6. E f f e c t o f v a r i o u s c o n c e n t r a t i o n s o f p r o c a i n e o n t h e s e c r e t i o n a n d r e t e n t i o n of r a d i o a c t i v e a l b u m i n b y liver slices. R a t liver s l i c e s w e r e i n c u b a t e d w i t h L - [ 1 4 C ] l e u c i n e f o r 9 m i n a n d w e r e t h e n " c h a s e d " f o r 9 0 m i n w i t h n o n - r a d i o a c t i v e L - l e u c i n e , 2 . 5 • 1 0 -4 M c y c l o h e x i m i d e a n d v a r i o u s c o n c e n t r a t i o n s o f p r o c a i n • . T h e r a d i o a c t i v e a l b u m i n r e t a i n e d in t h e slices o r s e c r e t e d i n t o t h e m e d i u m w a s d e t e r m i n e d b y i m m u n o p r e c i p i t a t i o n . T h e r e c o v e r i e s o f r a d i o a c t i v e a l b u m i n w e r e 8 0 0 0 c p m in t h e c o n t r o l a n d 6 4 3 1 c p m i n t h e h i g h e s t p r o c a i n e - t r e a t e d slices. T h e v a l u e s g i v e n are t h e m e a n a v e r a g e s o f 3 d e t e r m i n a t i o n s .

58 i n h i b i t o r y e f f e c t at 5 • 10 -2 M, and at all c o n c e n t r a t i o n s t e s t e d it caused b o t h an i n h i b i t i o n o f s e c r e t i o n a n d an a c c u m u l a t i o n o f the n o n - s e c r e t e d r a d i o a c t i v e a l b u m i n w i t h i n t h e liver slices. At 5 • 10 .3 M p r o c a i n e o n l y 10% of the a l b u m i n s y n t h e s i z e d was s e c r e t e d a n d t h e r e m a i n d e r was r e t a i n e d in the slices (Fig. 6).

Intracellular distribution of the non-secreted plasma proteins Since p r o c a i n e causes an i n h i b i t i o n of s e c r e t i o n a n d an a c c u m u l a t i o n of the s e c r e t o r y p r o d u c t s w i t h i n t h e cell (Fig. 6} and since we have p r e v i o u s l y s h o w n t h a t in c o l c h i c i n e - t r e a t e d a n i m a l s this a c c u m u l a t i o n o f the n o n - s e c r e t e d proteins o c c u r s in Golgi-derived s e c r e t o r y vesicles w i t h i n the h e p a t o c y t e , it was of i n t e r e s t to d e t e r m i n e in w h i c h cellular c o m p a r t m e n t the p r o c a i n e - i n d u c e d a c c u m u l a t i o n occurs. With this i n t e n t , liver slices were pulse-labelled with L-[14CJleucine f o r 9 m i n , w a s h e d t h o r o u g h l y with n o n - r a d i o a c t i v e leucine and f u r t h e r i n c u b a t e d in a chase m e d i u m , in the a b s e n c e or p r e s e n c e of 5 • 10 -'~ M p r o c a i n e or 5 • 10 -s M colchicine. B o t h the c o n t r o l and the d r u g - t r e a t e d slices c o n t a i n e d c y c l o h e x i m i d e in the " c h a s e " m e d i u m . This was d o n e to p r e v e n t f u r t h e r p r o t e i n s y n t h e s i s d u r i n g t h e chase p e r i o d . This b l o c k i n g of p r o t e i n synthesis in b o t h cases is n e c e s s a r y f o r c o m p a r i s o n o f the e f f e c t s of colchicine and p r o c a i n e on s e c r e t i o n since p r o c a i n e inhibits p r o t e i n s y n t h e s i s while colchicine d o e s n o t . A f t e r t h e 90 m i n chase p e r i o d , t h e m e d i u m a n d slices were s e p a r a t e d b y c e n t r i f u g a t i o n a n d the slices were w a s h e d t h o r o u g h l y , h o m o g e n i z e d and f r a c t i o n a t e d i n t o various cellular c o m p o n e n t s involved in the s e c r e t o r y process. R a d i o a c t i v e p r o t e i n s f r o m e a c h cell f r a c t i o n were p r e c i p i t a t e d with r a b b i t antis e r u m p r e p a r e d against r a t s e r u m p r o t e i n or against p u r i f i e d r a t a l b u m i n . As s h o w n p r e v i o u s l y (Table I, Fig. 6), p r o c a i n e i n h i b i t e d s e c r e t i o n into the med i u m and the i n h i b i t i o n o f s e c r e t i o n was a c c o m p a n i e d b y a c o n c o m i t a n t retent i o n o f the s e r u m p r o t e i n s w i t h i n t h e slices. In the c o n t r o l slices, 37.5% o f the s e r u m p r o t e i n s p r o d u c e d w e r e secreted into t h e m e d i u m while p r o c a i n e diminished this a m o u n t t o 24%. T h e c o n t r o l slices r e t a i n e d 6 2 . 5 % o f the r a d i o a c t i v e s e r u m p r o t e i n s p r o d u c e d while p r o c a i n e e n h a n c e d this r e t e n t i o n to 76%. In these e x p e r i m e n t s 6 g of slices, as c o m p a r e d t o 0.3 g in p r e v i o u s e x p e r i m e n t s , h a d to be used in o r d e r t o o b t a i n s u f f i c i e n t tissues f o r cell f r a c t i o n a t i o n . T h e use o f larger a m o u n t s of slices, a l t h o u g h i n c u b a t e d in c o r r e s p o n d i n g l y larger v o l u m e s o f i n c u b a t i o n m e d i u m , r e s u l t e d in c o n s i d e r a b l y less s e c r e t i o n of r a d i o a c t i v e s e r u m p r o t e i n s and p r o c a i n e was f o u n d t o h a v e a smaller i n h i b i t o r y e f f e c t t h a n w h e n smaller a m o u n t s o f slices were used ( C o m p a r e Fig. 6 a n d T a b l e III). U n d e r these conditions, h o w e v e r , p r o c a i n e caused a s u f f i c i e n t i n h i b i t i o n o f secretion and an increased a c c u m u l a t i o n w i t h i n t h e slices to allow us to m e a s u r e these changes. Colchicine, as s h o w n p r e v i o u s l y , also inhibits s e c r e t i o n in r a t liver slices and the p e r c e n t o f a l b u m i n s e c r e t i o n d r o p p e d f r o m 57% to 27% with colchicine treatm e r i t and the r a d i o a c t i v e a l b u m i n r e t a i n e d in the slices was raised f r o m 43% to 73% (Table I I I ) . When the slices w e r e f r a c t i o n a t e d i n t o various cellular f r a c t i o n s , the proc a i n e - t r e a t e d tissues, unlike c o l c h i c i n e , did n o t s h o w an a p p r e c i a b l e increase of r a d i o a c t i v e s e r u m p r o t e i n s in Golgi-derived s e c r e t o r y vesicles b u t it a p p e a r e d t h a t the r e t a i n e d s e r u m p r o t e i n s in t h e p r o c a i n e - t r e a t e d slices were dispersed a m o n g several cell f r a c t i o n s with s o m e a c c u m u l a t i o n in 8 0 0 × g and 12 100 X g

59 pellets and in the "soluble" cell fraction (Table III). The colchicine-treated slices had a greater accumulation of non-secreted protein than did procainetreated slices and although some increases in radioactive albumin could be seen in the 12 100 × g pellet, the rough and smooth endoplasmic reticulum and the soluble fraction, the largest percent increase occurred in the Golgi fraction with a 4-fold increase in Golgi fraction 3 and a 5-fold increase in Golgi fractions 1 and 2. Discussion These experiments show that some local anesthetics, and chlorpromazine, inhibit secretion in rat liver slices, but that, unlike colchicine, they do n o t appear to block secretion by hampering the discharge of Golgi-derived secretory vesicles. The experiments were performed with rat liver slices but there are several disadvantages to using this system. For instance, the results vary from experiment to experiment (see Tables I and II) and pulse-chase experiments show that after a 90 min chase period, substantial amounts of radioactive serum proteins are retained within the slices (Table III) while, in vivo, most of the serum proteins have been discharged from the cell [1]. Further comparison with in vivo studies [1] show that rat liver slices contain a larger percentage of radioactive serum protein in the soluble cell fraction, indicating the possibility of cellular damage during preparation. These studies, however, could not be performed in vivo, because the large dosage of local anesthetics required to block secretion is lethal to rats. The slices are useful, however, in that both colchicine and the local anesthetics inhibit secretion and thus their modes of action could be compared. The anesthetics, like colchicine, have no effect on the uptake of radioactive leucine, the uptake of radioactive phosphorous and its incorporation into intracellular nucleotide phosphates and phospholipids [ 1]. Unlike colchicine, however, they severely inhibit protein synthesis, as measured by the incorporation of radioactive leucine into trichloroacetic acid-precipitable proteins. It is possible that local anesthetics and colchicine may inhibit secretion by similar mechanisms since these drugs are known to induce structural changes in cells by affecting the cytoskeletal system of microfilaments and microtubules [2,3,26]. The detailed mechanisms of action of these drugs are, however, not y e t well understood and are still being debated. These experiments suggest that procaine, which has the least effect on protein synthesis of the local anesthetics tested, probably affects secretion at a different cellular site than does colchicine, since it does not appear to inhibit secretion at the level of exocytosis. It is still possible, however, that procaine and other local anesthetics may be acting like colchicine and are inhibiting secretion by affecting exocytosis, but that their other cellular effects, such as their known interaction with membranes [14] may mask this effect. For instance, in the experiments described in Table III, procaine may cause an accumulation of plasma proteins within the Golgi, but the procaine-treated Golgi-derived secretory vesicles, unlike the colchicinetreated ones, may be more susceptible to mechanical damage during homogenization. This is doubtful, however, since the percent increase of non-secreted plasma proteins in the soluble cell fraction from procaine-treated slices is not larger than that from colchicine-treated slices.

60 Due to the multiple effects of the local anesthetics and n o t being able to pinpoint at which step or steps in the secretory pathway they are acting, it is n o t possible at present to attribute their inhibitory effect on plasma protein secretion to their effect on any particular cellular function or structure. These experiments do suggest, however, t h a t the local anesthetics inhibit secretion at different cellular sites than does colchicine and that one cannot at present ascribe their inhibitory effect to their antimicrotubular activity. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

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