the effects of γ-hexachlorocyclohexane on amylase secretion and inositol phospholipid metabolism in...

Biochimica et Biophysica Acta 844 (1985) 149-157 149 Elsevier

BBA 11404

T h e e f f e c t s o f y - h e x a c h l o r o c y c l o h e x a n e o n a m y l a s e s e c r e t i o n an d inos i to l

p h o s p h o l i p i d m e t a b o l i s m in m o u s e p a n c r e a t i c ac in i

M i c h a e l F. C r o u c h * a n d M i c h a e l L. R o b e r t s

Department of Physiology, University of Adelaide, Adelaide, SA 5000 (Australia)

(Received April 6tb, 1984) (Revised manuscript received October 4th, 1984)

Key words: Hexachlorocyclohexane; Amylase secretion; Inositol phospholipid; (Mouse acini)

Dispersed mouse and guinea-pig pancreatic acini were used to examine the effects of the inositol analogue, y-bexachlorocyclohexane (lindane) on agonist-stimulated amylase secretion. Secretion from mouse acini in response to carbachol and cholecystokinin octapeptide (CCK-8) was reduced by lindane. Similarly, amylase release from guinea-pig acini stimulated by carbachol was abolished by lindane. These acini, however, still remained responsive to dibutyryl-cAMP with only a slightly diminished secretion to this agent. Inositol phospholipid synthesis and hydrolysis was stimulated in mouse acini by both carbachol and CCK-8. Although hydrolysis of these lipids in response to CCK-8 was reduced by only 18%, stimulation of inositol phospholipid synthesis by either agonist was abolished by lindane. Dose-response curves for inositol phospholipid synthesis stimulated by carbachol and CCK-8 in mouse acini were biphasic and superimposable with those of amylase secretion. In contrast, the dose-response curve for phosphoinositide hydrolysis was sigmoid and clearly separable from that of synthesis. Reducing the external Ca 2 + concentration caused the dose-response curves for carbachol- and CCK-8-induced inositol phospholipid synthesis to be displaced to the right, as has been observed for amylase secretion. A23187 was also found to induce amylase secretion and inositol phospholipid synthesis, and both of these responses were inhibited by lindane. Amylase secretion and inositol phospholipid synthesis may, therefore, be closely related events in the exocrine pancreas. Lindane may provide a valuable tool with which to determine the role of inositol phospholipid metabolism in stimulus-response coupling.

Introduction

Occupation of certain cell surface receptors by agonists has been shown to initiate the breakdown and resynthesis of inositol phospholipids. This effect has been observed in response to agonists which utilize Ca 2+ as their second messenger and it has been suggested that this receptor-activated response is somehow responsible for allowing Ca 2 ÷ into the cell [1-3].

* To whom correspondence should be addressed. Abbreviations: CCK-8, cholecystokinin octapeptide; Hepes, 4- (2-hydroxyethyl)-l-piperazineethanesulphonic acid.

In some cells, however, hydrolysis of inositol phospholipids does not appear to be involved in controlling Ca 2 ÷ entry [4-6]. In addition, there is evidence for roles of this lipid response in cell function other than causing Ca 2÷ entry (e.g., Ref. 7). One approach to clarifying the role that the inositol phospholipid response plays in cellular functions would be by the use of specific inhibitors of inositol phospholipid turnover, and there is evidence that analogues of inositol can be used for this purpose. Using the myo-inositol analogue, 7- hexachlorocyclohexane (lindane), Hokin and Brown [8] found a potent inhibition of acetylcho- line-stimulated phosphatidylinositol formation in

0167-4889/85/$03.30 © 1985 Elsevier Science Publishers B.V.

150

guinea-pig cerebral cortex. Fisher and Mueller [9] described an inhibition of both cell growth and phosphatidylinositol synthesis by this analogue in phytohaemagglutinin-stimulated lymphocytes. Cell growth is, however, a complex response, with many processes separating the stimulus and growth. Lin- dane has not been used to study cell function in cells which exhibit a closer temporal relationship between inositol phospholipid turnover, Ca 2+ fluxes and response.

We have chosen the exocrine pancreas to study the action of lindane. Mouse acini possess a number of receptors which stimulate secretion by in- creasing Ca z+ influx [10,11]. In the guinea-pig pancreas, amylase secretion can be stimulated through a cAMP-dependent pathway in addition to the Ca2+-dependent pathway [12]. Since cAMP does not induce inositol phospholipid turnover (for review see Ref. 2), we have been able to examine the specificity of action of lindane in its effects on exocrine secretion.

In the course of this work it became clear that agonist-stimulated inositol phospholipid metabolism is more complex than previously described. In addition to the hydrolysis of inositol phospholipids, which is CaZ+-independent [13], there is an agonist-induced synthesis of inositol phospholipids which occurs independently of the hydrolysis.

Materials and Methods

Preparation of mouse pancreatic acini. Isolated mouse pancreatic acini were prepared essentially according to the method of Williams et al. [14]. Acini were dissociated using collagenase and resuspended in 25 ml polycarbonate flasks with Krebs-Henseleit bicarbonate solution of the following composition (mM): NaC1, 118; KC1, 4.7; NaH2PO4, 1.2; glucose, 14; NaHCO 3, 25; and CaCI 2, 1.28. This incubating solution was changed in later experiments, to improve acinar responsive- ness, by substituting the NaHCO 3 for 10 mM Hepes (Krebs-Henseleit-Hepes buffer). The buffer used in each series of experiments is detailed in the figure legends. During all experiments, acinar samples were kept at 37°C and were gassed continu- ously with either 95% 02 /5% CO 2 when the buffer used was Krebs-Henseleit bicarbonate, or 100% 02 when Krebs-Henseleit-Hepes was the buffering

solution, and samples were shaken at 60 cycles/min. When samples were treated with lindane, it was added in dimethyl sulphoxide (5 /L1/ml). To those samples not treated with lindane, dimethyl sulphoxide was added alone. In all experiments described, the lindane used was of com- mercial grade and was always at a final concentration of 0.1 mM.

Preparation of guinea-pig pancreatic acini. Iso- late guinea-pig acini were prepared by the method of Peikin et al. [15]. Acini were dispersed with pure collagenase (0.18 mg/ml) and acinar samples were handled in the same manner as were mouse acini.

Amylase secretion from acini. Following a 75 min incubation with either dimethyl sulphoxide or lindane (or 35 min in later experiments, as this was found to be equally effective), acini were centrifuged at 50 × g for 4 min and resuspended in 20 ml of fresh buffer with either dimethyl sulphoxide or lindane. Aliquots (1.8 ml) of acini were placed in a number of polycarbonate flasks and 0.2 ml of agonist was added to each. After 30 min, a 0.5 ml sample was taken from each flask and this was used to determine the amount of amylase released during the incubation period as a percentage of the total acinar amylase present at the start of the incubation. Amylase was determined using Re- mazol brilliant blue-labelled starch [16].

Agonist-stimulated [3H]inositol phospholipid synthesis. Isolated acini were divided into 10-ml samples and, when appropriate, to one was added lindane and to the other dimethyl sulphoxide alone. After 45 rain they were centrifuged for 4 min at 50 × g and each pellet was resuspended with 15 ml of buffer containing lindane or dimethyl sulphoxide as appropriate. Samples of the acini (880 /~1) were dispensed into 5-ml polypropylene vials and to each was added 20 /~1 of myo-[2-3H]inositol, giving a final concentration of 0.7/~Ci/ml. After 30 min, 0.1 ml of agonist or buffer alone was added and the incubation continued for a further 30 min. Samples were precipitated with 200 t~l of trichloroacetic acid (50% w / v containing 10 mM carrier myo-inositol) at the end of the incubation, diluted with 3 ml of buffer and rapidly filtered through G F / B glass-fibre filters using a filtration manifold. Filters were then rinsed with a further 9 ml of buffer, placed in 5-ml scintillation vials and counted in 3.5 ml of scintillation cocktail using a

151

Packard scintillation counter. Cellular uptake of myo-[2-SH]inositol. Total

cellular uptake of myo-[2-3H]inositol was measured using a protocol similar to that for phospholipid synthesis except that, following the incubation with or without agonist, samples were not protein-precipitated but were immediately diluted with 3 ml of buffer and rapidly filtered, washed and counted as for synthesis experiments.

Agonist-induced hydrolysis of [3H]inositol phospholipids. In order to measure stimulated hydrolysis of inositol phospholipids in mouse acini, pre-labelling of these lipids using myo-[2-3 H]inositol was carried out either in vitro or in vivo. In vitro labelling required lengthy incubations but it proved to be a satisfactory method for most experiments. In those experiments designed to determine the effects of lindane on hydrolysis, however, cell death was abnormally high following the incubation with lindane, and this dictated a change of labelling protocol for these experiments. Labelling in vitro was carried out as described previously [17] by intraperitoneal injection of 15 /~Ci per mouse of myo-[2-3H]inositol. Acini were obtained from the prelabelled pancreas as described above.

In vitro labelling was carried out by first prein- cubating acini for 30 min with myo-[2-3H]inositol (4/~Ci/ml) in 5 ml of buffer. Synthesis of [3H]inositol phospholipids was then stimulated for 30 min by the addition of carbachol (1.0 ~M) and after this time most of the free extracellular myo- [2-3H]inositol was removed by two 5 ml washes of the acini with buffer. Acini were resuspended with buffer containing atropine (10 /LM). These in vitro-labelled acini were then allowed to incubate in this buffer for 30 min prior to stimulation of inositol phospholipid hydrolysis. During the equivalent period for in vivo-labelled tissue, acini were incubated with either lindane or dimethyl sulphoxide.

Measurement of inositol phospholipid hydrolysis in response to CCK-8 was carried out using the technique described by Berridge et al. [18]. In the presence of Li, inositol-l-phosphatase is inhibited and one can use the accumulation of [3H]inositol phosphates as a direct measure of inositol phospholipid hydrolysis. In accordance with the results of Berridge et al. [18], inositol-l-phosphatase was maximally inhibited in the presence of 10 mM

LiC1 (results not presented). Measurement of muscarinic receptors on mouse

acini. Acini were preincubated for 35 min in buffer containing either lindane or dimethyl sulphoxide, washed and resuspended with lindane or dimethyl sulphoxide as appropriate. Samples of acini (880 ~1) were added to polypropylene vials and 100 ~1 of atropine (100 #M final concentration) or buffer were added. After 15 min, 20 ~1 of [3H]quinuclidinyl benzylate was added (final concentration 3 nM). Incubations were terminated after 2 h by dilution with 3 ml of 0.1 M phosphate buffer followed by rapid filtration through GF/B glass- fibre filters. The filters were washed with 9 ml of phosphate buffer and samples were counted as described above. Specific binding to muscarinic receptors represented the difference in counts between samples with and without atropine.

Assay of DNA content of acinar samples. The DNA content of acinar samples was determined using the method of Labarca and Paigen [19].

Statistics. Statistical comparisons of data employed Student's t-test, except for results in Table II, which were compared by an analysis of variance.

Chemicals. Soybean trypsin inhibitor type 1-S, bovine serum albumin (fraction V), DNA, lindane, atropine, carbachol, myo-inositol, Hepes and Re- mazol brilliant blue were obtained from Sigma Chemical Co. (U.S.A.). Collagenase was from Worthington Biochemicals. Hoechst dye No. 33258 was from Calbiochem-Behring Corp. (U.S.A.). myo-[2-3H]Inositol (16 Ci/mmol) and [3H]- quinuclidinyl benzylate were purchased from The Radiochemical Centre, Amersham (U.K.). Scintil- lation cocktail (E299) was from Packard and glass-fibre filters from Whatman (U.K.). CCK-8 was a gift of E.R. Squibb and Sons (New Jersey).

Results

Effect of lindane on amylase secretion from pancreatic acini

Lindane inhibited amylase secretion induced by carbachol and CCK-8 (Table I) and A23187 (Ta- ble III) from mouse pancreatic acini. Carbachol- induced amylase secretion from guinea-pig acini was also abolished by lindane, whereas that stimulated by dibutyryl-cAMP was reduced by only 35% (Table II).

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TABLE I

THE EFFECT OF LINDANE ON AGONIST-STIMU- LATED AMYLASE SECRETION FROM MOUSE PAN- CREATIC ACINI

Mouse acini were pre-incubated with or without lindane (0.1 mM) for 75 min and to acinar samples was then added either carbachol (10 6 M), CCK-8 (10 l0 M) or buffer alone. After 30 min, an aliquot was taken from each sample and assayed for amylase secreted as a percentage of the total cellular amylase. Each value is the mean ± S.E. of 11 samples from four separate experiments. The buffer system used was Krebs-Henseleit bicarbonate medium.

Agonist Amylase secreted above basal

Controlacini Carbachol 5.20±0.68 CCK-8 4.69±0.60

Lindane-treated Carbachol 1.21±0.37 ~ acini CCK-8 1.67±0.23 a

Significantly less than stimulated control acini (P < 0.01).

Effect of lindane on inositol phospholipid hydrolysis H y d r o l y s i s o n p h o s p h o i n o s i t i d e s in m o u s e ac in i

s t i m u l a t e d b y C C K - 8 (0.1 /~M) was r e d u c e d b y

18% ( P < 0 .01) f o l l o w i n g l i n d a n e t r e a t m e n t . L in -

TABLE II

THE EFFECT OF LINDANE ON AMYLASE SECRETION FROM GUINEA-PIG PANCREATIC ACINI IN RE- SPONSE TO CARBACHOL AND DIBUTYRYL-cAMP

Isolated guinea-pig acini were pre-incubated with or without lindane (0.1 mM) for 35 min and then to acinar samples was added either carbachol (10- s M), dibutyryl cAMP (10 3 M) or buffer alone. After 30 min, an aliquot was taken from each sample to determine the amount of amylase secreted as a percent of total cellular amylase. Acini were maintained in Krebs-Henseleit-Hepes buffer containing 5 mM theophylline. Results are the means + S.E. of three separate experiments with triplicate samples in each. Analysis of variance revealed that the responses to both carbachol and dibutyryl cAMP were reduced by lindane (P < 0.01) but that the effect of lindane on the dibutyryl cAMP-induced secretion was smaller than on the carbachol-induced secretion (P < 0.01).

Agonist Amylase secretion above basal

Control acini Carbachol 8.57 + 0.85 dibutyryl cAMP 8.99 ± 0.83

Lindane-treated Carbachol - 0.45 ± 0.21 acini dibutyryl cAMP 5.43 _+ 0.52

TABLE III

THE EFFECT OF LINDANE ON AMYLASE SECRETION AND PHOSPHOINOSITIDE SYNTHESIS IN RESPONSE TO A23187 IN MOUSE PANCREATIC ACINI

Acini were preincubated with or without lindane (0.1 raM) for 35 min and then stimulated with A23187 (1.0 t~M) for 30 rain. Amylase secretion was measured using Remazol brilliant blue- labelled starch and inositol phospholipid synthesis by the incorporation of mvo-[2- 3 H]inositol into trichloroacetic acid-precipitable phospholipid. Values are the means + S.E. of three experiments for each response. The buffer system used was Krcbs- ttenseleit bicarbonate medium. Samples used to determine [3H]inositol phospholipid formation contained 30-50 ~g of DNA each.

Addition Amylase [ 3 H]Inositol secreted phospholipid (% total) formation

(dpm/~g DNA)

Controlacini A23187 18.0±2.2 17.1+1.1 0 5.5±1.2 6.2+0.2

Lindane-treated A23187 10.2±0.9 ~ 5.6±0.8 ~ acini 0 5.9±0.5 3.1+0.5 ~

Significantly less than corresponding control value ( P < 0.01 ).

d a n e - t r e a t e d a n d c o n t r o l s a m p l e s s h o w e d 12.01 _+

0 .32 a n d 9.79 _+ 0.47 d p m / / ~ g D N A , re spec t ive ly ,

o f [ 3 H ] i n o s i t o l p h o s p h a t e f o r m a t i o n .

Effect of lindane on [3H]quinuclidinyl benzylate binding to mouse pancreatic acini

T h e t o t a l b i n d i n g of [ 3 H ] q u i n u c l i d i n y l benzy l -

a t e to m o u s e ac in i was u n a f f e c t e d b y l i n d a n e

( T a b l e IV). N o n s p e c i f i c b i n d i n g , h o w e v e r , was re-

d u c e d in t he se s amp le s .

Inositol phospholipid synthesis and the effect of lindane

[ 3 H ] I n o s i t o l p h o s p h o l i p i d s y n t h e s i s was m e a -

s u r e d as t he 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 b l e r a d i o -

a c t i v i t y of m o u s e p a n c r e a t i c ac in i . V e r i f i c a t i o n

t h a t the l a b e l l e d p h o s p h o l i p i d s we re in fac t p h o s -

p h o i n o s i t i d e s was d o n e u s i n g t h e ac id i f i ed s o l v e n t

t e c h n i q u e of F a i n a n d B e r r i d g e [20] for e x t r a c t i o n

a n d T L C o n s i l ica gel p l a t e s to s e p a r a t e l ip ids .

U s i n g a C H C 1 3 / m e t h a n o l / 4 M N H 4 O H ( 9 : 7 : 2,

v / v ) s o l v e n t sys t em, r a d i o a c t i v i t y was l oca l i z ed to

t h e i nos i t o l p h o s p h o l i p i d s w i t h the c o n t r i b u t i o n s

o f p h o s p h a t i d y l i n o s i t o l , p h o s p h a t i d y l i n o s i t o l 4-

153

TABLE IV

THE EFFECT OF LINDANE ON [3H]QUINUCLIDINYL BENZYLATE BINDING TO MOUSE PANCREATIC ACINI

Following a 30 min incubation with lindane (0.1 mM in 0.5 v / v dimethyl sulphoxide) or dimethyl sulphoxide alone, 1 ml acinar samples were exposed to [3H]quinuclidinyl benzylate (3 mM) in the absence (total binding) or presence (nonspecific binding) of 100 ~M atropine. Samples were then filtered, washed and counted. Specific binding to muscarinic receptors represents the difference between total and nonspecific binding of [3H]quinuclidinyl benzylate ([3H]quinuclidinyl benzylate). Each value is the mean + S.E. of two experiments each with triplicate samples. Each acinar sample contained 30-50/~g of DNA. DMSO, dimethyl sulphoxide.

DMSO Lindane t-test

Total [ 3 H]quinuclidinyl benzylate bound dpm//xg DNA)

Nonspecific [ 3 H]quinuclidinyl benzylate bound ( d p m / # g DNA)

Specifically bound [3 H]quinuclidinyl benzylate (dpm//~g DNA)

500_+34 425_+4 P < 0.5

227_+ 7 79_+ 6 P < 0.001

273_+39 346_+4 P < 0.5

monophosphate and phosphatidylinositol 4,5-bis- phosphate being 84.3 + 1.3, 1.1 + 0.2 and 2.1 + 0.3%, respectively. No other radioactive peaks were observed.

Inositol phospholipid synthesis was stimulated more than 2-fold by carbachol in mouse pancreatic acini (Table V). Lindane reduced the rate of inositol phospholipid synthesis in unstimulated cells and completely inhibited the effect of carbachol on this synthesis (Table V). Virtually identical results were obtained when A23187 (Ta- ble III) or CCK-8 (0.1 nM) (detailed results not

presented) was used as the agonist, with stimulated phospholipid synthesis being blocked by lindane.

Lindane could achieve this inhibition in a number of ways, including by interfering with the cellular uptake of myo-[2-3H]inositol across the plasma membrane or by blocking synthesis di- rectly at a cytoplasmic site, probably the endo- plasmic reticulum [21,22]. These two possibilities were examined simultaneously by comparing total cellular myo-[2-3H]inositol uptake with the pro- portion of it incorporated into [3H]inositol phospholipids in stimulated and unstimulated acini.

TABLE V

THE EFFECT OF LINDANE (10 -4 mol/ l ) ON THE INCORPORATION OF myo-[2-3H]INOSITOL INTO [3H]INOSITOL PHOSPHOLIPID AND ON TOTAL UPTAKE OF myo-[2-3H]INOSITOL INTO ACINAR CELLS IN THE PRESENCE AND ABSENCE OF CARBACHOL (10 -6 tool/l).

Values represent the means + S.E. of eight samples in three separate experiments. The values for free cellular myo-[2-3H]inositol were obtained by subtracting the mean for the incorporated myo-[2-3H]inositol from the appropriate mean for total uptake. The buffer system for these experiments was Krebs-Henseleit bicarbonate medium. Samples contained 30-50/~g of DNA each.

Control acini Lindane-treated acini

Unstimulated Carbachol Unstimulated Carbachol

Synthesized [ 3 H]inositol phospholipids (dpm//~g DNA) 11.1 + 1.4

myo-[2- 3 H]lnositol uptake ( d p m / # g DNA) 22.5 + 1.8

Free cellular myo-[2- 3 H]inositol (dpm/~ g DNA) 11.1

28.2±2.2 a 7.0±1.2 b 7.1+1.2 b

32.3±2.0 ~ 11.8±0.6 b 14.0±0.9 b

4.1 4.8 6.9

a Significantly greater than unstimulated control acini ( P < 0.01). b Significantly less than unstimulated control acini (P < 0.05).

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Unstimulated control acini accumulated myo-[2- 3H]inositol over the incubation period and about 50% of this was incorporated into inositol phospholipids (Table V). Acini stimulated with carbachol showed increased uptake and, pro- portionately, an even greater stimulation of inositol phospholipid synthesis (Table V). In the presence of lindane, acini accumulated myo-[2- 3H]inositol but at a slower rate than untreated acini, indicating that lindane reduced but did not eliminate myo-[2-3H]inositol uptake (Table V). A reduction of inositol uptake has been reported to be produced by a variety of sugars in hamster small intestine [23] and by glucose and scyllo-inositol in rat liver cells [24]. Carbachol caused no stimulation of [3H]inositol phospholipid synthesis following lindane treatment despite having a free cellular myo-[2-3H]inositol level greater than untreated samples (Table V).

Dose-response relationships of amylase secretion and inositol phospholipid synthesis and hydrolysis in mouse acini

A comparison of dose-response curves of inositol phopsholipid synthesis and hydrolysis stimulated by CCK-8 revealed that these curves differed both in shape and in the range over which

z 15o

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Fig. 1. A comparison of CCK-8-induced inositol phospholipid synthesis (solid line) and hydrolysis (broken line) in mouse pancreatic acini. Synthesis was measured as the trichloroacetic acid-precipitable radioactivity of acini after 30 min with agonist and myo-[2-3 H]inositol. Hydrolysis was assayed by the increase in [ 3 H]inositol phosphate release from in vitro-labelled [ 3 H]inositol phospholipids. The protocol employed the use of Li ÷ to amplify inositol phosphate accumulation, Results are the means -t- S.E. of 4-10 samples from 2-5 experiments. The buffer used in these experiments was Krebs-Henseleit-Hepes.

c~ 16 tO

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CD

---I"--- £ I CO ~ ~O

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< 50

2 H G 7

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GARSACHOL ( - l o 9 N)

Fig. 2. Dose-response curves for carbachol-stimulated amylase secretion (solid line) and inositol phospholipid synthesis (broken fine) in mouse pancreatic acini. Synthesis of inositol phospholipids was measured as the acid-precipitable radioactivity following incubation with carbachol and myo-[2-3H]inositol. Acinar samples were maintained in Krebs-Henseleit-Hepes buffer. Results are the m e a n s i S , E. of three experiments for both lipid synthesis and amylase secretion.

CCK-8 was effective (Fig. 1). The dose-response curve for phosphoinositide synthesis was biphasic, with maximum synthesis occurring at 0.1 nM

2O

]8

s < 16

]4

]0 W

CJ Ld O~ ~'

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CCK-8 (-]o9 M)

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Fig. 3. Dose-response curves for CCK-8-induced amylase secretion (solid line) and inositol phospholipid synthesis (broken line) in mouse pancreatic acinL [3H]lnositol phosphofipids were assayed as the acid-precipitable radioactivity following stimulation with CCK-8 in the presence of myo-[2-3H]inositol. Incubations were performed in Krebs-Henseleit-Hepes buffer and represent the means±S.E, of four experiments for phosphoinositide synthesis and eight experiments for amylase secretion.

155

150

~ r ct:

o I00 r 5 2 g o=~ 50 CL

d

(=3 z S II i0 9 8

CCK-8 (-Io S M)

Fig. 4. [3H]Inositol phospholipid synthesis in response to CCK-8 over a 30 min period in the presence of 1.28 mM CaCI 2 (solid line) and 0.1 mM CaC12 (broken line). Acini were incubated in Krebs-Henseleit-Hepes buffer containing myo-[2- 3 H]inositol with or without CCK-8. Results are the means + S.E. of six samples in two separate experiments involving 0.1 mM CaCI/ , and 12 samples in four experiments when using 1.28 m M CaCI 2.

CCK-8. At 10 nM CCK-8 the response was near unstimulated values (Fig. 1). Hydrolysis of the lipid followed a sigmoid curve, with a near maximal response at 10 nM CCK-8 (Fig. 1).

The dose-response curves of amylase secretion and inositol phospholipid synthesis induced by carbachol (Fig. 2) and CCK-8 (Fig. 3) in the mouse were found to be coincident with respect to agonist potency. Although CCK-8 appears to pro- mote more amylase secretion than carbachol in these experiments, this is largely explained by the greater release of amylase seen in the unstimulated samples in the experiments using CCK-8.

Reducing the external Ca 2+ concentration of the medium from 1.28 to 0.1 mM induced shifts of the dose-response curve for the synthesis of inositol phospholipid stimulated by either CCK-8 (Fig. 4) or carbachol (results not shown). The inhibition of synthesis at higher concentrations was reduced for both agonists, the peak synthesis occurring at higher agonist concentrations, and synthesis in response to submaximal doses was less than found in the higher-Ca 2 +-containing medium.

Discussion

The ability of lindane to inhibit amylase secretion from the exocrine pancreas in response to

agents which mobilize Ca 2+ (Tables I, II and II) but to a much lesser extent when stimulated by an increase in the intracellular cAMP level (Table II) showed that this compound was not acting as a cytotoxin, but was specific in its effects. A number of hydrophobic compounds, such as procaine [25] and TMB-8 [26], have been shown to have receptor-blocking qualities. This did not appear to ex- plain the present results, since [3H]quinuclidinyl benzylate binding to muscarinic receptors was not reduced by lindane (Table IV).

It has been proposed that hydrolysis of inositol phospholipids is involved in the stimulus-response pathways for CaZ+-dependent secretion [1-3]. Consequently, the effect of lindane on the agonist-stimulated turnover of these lipids was investigated. Lindane reduced only marginally the CCK-8-induced inositol phospholipid hydrolysis. Creba et al. [27] similarly found a lack of effect on inositol phospholipid hydrolysis of another inositol analogue, delta-hexachlorocyclohexane, in the hepatocyte.

In contrast, to hydrolysis, synthesis of inositol phospholipids in response to carbachol was abolished by lindane (Table V). The reduced cellular uptake of myo-[2-3H]inositol could not account for all of this inhibition, since carbachol reduced the amount of free myo-[2-3 H]inositol, by stimulat- ing inositol phospholipid synthesis, in control tis- sues to a level below that observed in lindane- treated acini (Table IV). This indicates that the synthesis mechanism was affected at some point.

Inositol phospholipid synthesis is often consid- ered to represent the compensatory mechanism to replenish stores depleted through agonist-stimulated hydrolysis. We investigated this in mouse acini by comparing CCK-8 dose-response curves of hydrolysis and synthesis of [3H]inositol phospholipids. At agonist concentrations which stimulate detectable synthesis, no inositol phospholipids hydrolysis was apparent (Fig. 1). Also at high CCK-8 concentrations, at which hydrolysis was near maximal, synthesis of phosphoinositides was inhibited and approached control levels (Fig. 1). These results indicate that two separable inositol phospholipid metabolic events can occur in the mouse pancreas following receptor activation, those of hydrolysis and synthesis, and that at high agonist concentrations the hydrolysed inositol

156

phospholipid pool is not simultaneously re- plenished by stimulated resynthesis. An increase in the stimulated synthesis of phosphoinositides through the de novo pathway has been reported in rat [28,29], human [28] and pigeon pancreas [30], and this mechanism may also be responsible, at least in part, for the stimulated synthesis in the mouse pancreas. A similar dissociation of dose-response curves for synthesis and hydrolysis of phosphoinositides can be seen in the rat parotid when comparing the studies of Weiss and Putney [31] and Michell et al. [32].

The observation that A23187 can elicit both amylase secretion and inositol phospholipid synthesis (Table III) indicates that each can be activated by Ca 2 +-influx into the cells. The rise in intracellular Ca 2+ concentration stimulated by carbachol and CCK-8 [7,11] is probably the mechanism by which these agents stimulate inositol phospholipid synthesis. The blockade by lindane of both amylase secretion (Table III) and phosphoinositide synthesis (Table III) produced by A23187 again suggests an effect of lindane at a post-receptor site.

Since both synthesis of inositol phospholipids and amylase secretion were inhibited by lindane in mouse acini, we looked further to see whether a relationship existed between these two responses. Dose-response curves for both responses stimulated by either CCK-8 (Fig. 3) or carbachol (results not shown) were biphasic, and for each agonist these two curves were virtually superimposable (Fig. 3). The stimulation of amylase secretion with low concentrations of agonist [10] and the inhibition of secretion with high concentrations [33-35] are both Ca2+-dependent events. This reduction in secretion can be attenuated if the external Ca z+ concentration is reduced [33,34]. This effect was also observed for inositol phospholipid synthesis induced by CCK-8 (Fig. 4) and carbachol (results not shown) when the medium C a 2+ w a s reduced from 1.28 to 0.1 mM. These comparisons of dose-response curves suggested that agonist-stimulated amylase secretion and inositol phospholipid synthesis are related Ca2+-depen - dent events in the mouse pancreas.

The present results show that lindane acts specifically to inhibit Ca2+-mediated amylase secretion and inositol phospholipid synthesis, and

not the cAMP-mediated amylase secretion. These effects of lindane do not appear to be due to a reduction in inositol phospholipid hydrolysis. Fur- ther experiments are being undertaken to determine whether inositol phospholipid synthesis is essential for Ca 2+-mediated exocrine secretion or whether these are simultaneous CaZ+-dependent events, with lindane interrupting a common pathway.

Acknowledgements

We would like to thank Dr. M.A. Ondetti for his generous gift of CCK-8. This study was sup- ported by the National Health and Medical Re- search Council of Australia.

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