Evaluation of the role of calcium in cytotoxic injury in isolated rat pancreatic acini

Vol. 139, No. 2, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

September 16, 1986

Pages 530-537

EVALUATION OF THE ROLE OF CALCIUM IN CYTOTOXIC INJURY IN ISOLATED RAT PANCREATIC ACINI

A.P.N. M~um~a~, 1'2 G.D. Vesenka?1~ M.A. Dubick, '~ and M.C. Geokas --5 IEnzymology Research Laboratory, Department of Medicine, Veterans Administration Medical Center, Martlnez, California 94553

2Departments of Biological Chemistry

and3Medicine,

University of California, Davis,Callfornia 95616 Received July 24, 1986

The role of extracellular Ca 2+ in pancreatic acinar membrane damage (cellular injury) by nicotine, membrane-active agents (mellitin, snake venom and Ca ionophore A23187) and secretagogues (CCK-8 and secretin) was investigated.Freshly isolated dispersed pancreatic acini from 18 h fasted adult rats were incubated wit~. one of the aforementioned agents, in the absence and presence o~ICa -T. Cellular injury was assessed by measuring the release of pulse-labeled ~'Cr and LDH. In addition, release of amylase, try~$ sinogen and chymotrypsinogen was also de~@rmined. In the absence of Ca nicotine (6 mM) caused a profound release of J'Cr and LDH as well as amylase, trypsinogen and chym~rypsinogen from the isolated pancreatic aeini. Release of these enzymes and ~-Cr decreased sharply w i t ~ a d d i t i o n of increasing concentrations (0.25-5 mM) of Ca ~ . Release of ~'Cr and amylase by snake venom (5~+ug/ml) was found to be 100 and 25% higher, respectively,in the absence of Ca ~ than in its presence. On the other ha~@, the Ca ~ ionophore A23187 (7 ~g/ml) was foundg~o be effective in releasing ~- Cr and amylase only in the presence of Ca ~ . CCK-8, (0.25nM), secretin (I~M) and mellitin (0.5 ~g/ml) a l t h o u ~ significantly stimulated amylas~Isecretion (225-350%) in the presence of Ca- ,none of the agents induced ~'Cr r ~ e a s e from acini, either in the absence or in the+presence of extracellular Ca . It is concluded that the extracellular Ca- plays no specific role in cytotoxic injury in isolated pancreatic acini. © 1986 AcademicPress, Inc.

Over the years a variety of evidence has appeared to indicate that extracellular 6).

Ca 2+ plays a critical role in the initiation of toxic cell death (I-

Utilizing primary hepatocyte cultures, Sehanne

that

a

number

of

et

al

(7)

demonstrated

membrane-active toxins were cytotoxic only in presence of 2+

high concentrations of extracellular Ca others

by

demonstrating

a

dependence

injury to hepatocytes in culture

by

on

carbon

0006-291X/86 $1.50 Copy)'ight © 1986 by Academt~' Press, lnc'. All rights of reproduction in any jorm reserved.

This was subsequently supported by

530

extracellular Ca 2÷ for cytotoxic tetrachloride

(CCI 4)

and

2,4-

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 139, No. 2, 1986

dinitrophenol critical et

(8,9).

involvement

ai(11)

Although these and other similar observations of extracellular

Ca 2+ in cytotoxic injury

- or ethylmethane

dependent upon extra-cellular

sulfonate - induced Ca 2+. Furthermore,

relationship between the concentrations

of

extracellular

caused by either CCI 4 or t-butylhyperoxide

cytes.

This controversy, hepatocytes,

cellular

the CCI 4 -,

injury

was

Ca 2+

and

of

using

in freshly isolated hepato-

and the fact that previous

prompted us to reinvestigate

studies

have

primarily

the role of extracellular

dispersed

stimuli.

Utilizing freshly iso-

pancreatic acini, we have examined the involvement

of extra-

cellular Ca 2+ in initiating membrane damage by a variety of toxic agents litin,

snake

The

isolated pancreatic acini is that they retain their high

metabolic acvity and res,pond to physiological lated

not

cellular

Ca 2+ in cytotoxic injury in freshly isolated dispersed pancreatic acini. advantage

Smith

Smith and Sandy (12) found no

injury

utilized

(7,10),

reported that at least in freshly isolated hepatocytes

bromobenzene

suggest a

venom and Ca 2÷ ionophore A23187) and nicotine,

tuent of tobacco, which is known to affect the structural

the main consti-

and functional

perties of a number of organs including the exocrine pancreas

(mel-

pro-

(13,14).

MATERIALS AND METHODS

Dispersed papncreatic acini were isolated from 18 h fasted adult male Sprague- Dawley rats (175-200g) with a slight modification (13,14) of the procedure described by Williams et al (15). Routinely, acini isolated from I 1.2g of pancreatic tissue were suspended in 10 ml of p r e w a r m ~ (37°C) oxygenated HEPES-buffered Ringer (HR-buffer) containing 1.2~ mM Ca ~ and incubated at 37°C for 45 min in the presence of 2 0 U ~ 51Cr (Sodium chromate, Amersham). At the end of this incubation period the Cr-labeled acini were recovered by l o w - s p e ~ centrifugation (100 xg for 3 min), washed twice with HR-buffer devoid of Ca- and finally resuspended in 45-50 ml of Ca 2÷ - devoid HR-buffer. A small aliquot from each preparation was mixed with an equal volume of 0.4% trypan blue in HR-buffer, and the exclusion of the dye by the cells, as an essessment of cellular viability, was tested. Preparations containing over 95% viable cells were utilized. In all experiments I ml aliquots of cell suspension ~re incubated at 37°C for 30 min in the absence (basal) and presence of Ca as stated in the legends to figures. The basal incubation also contained 0.1 mM EGTA. The agents to be tested were added at the start of the 30 min incubation period. The reactions were terminated by centrifugation at 2000 x g for ~9 min. Ali ~ quots of the 2000 x g supernatant (medium) were counted for ~-Cr, and also assayed for amylase (16), trypsinogen (14), chymotrypsinogen (17) and lactate dehydrogenase (LDH) as described previously (13,14). The 2000 x g pellet (cells) was suspended in I ml of dispersing buffer ( 0 . 0 1 M Tris-HCI, pH 7.5

531

Vol. 139, No. 2, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

0.1% bovine serum albumin - O.05M CaCI~ - 0.1% Triton - X-100) and sonicated for 20 min, and an aliquot (0.1ml) was counted for ~-Cr. The rest of the sonicated material was centrifuged and the supernatant was assayed f o r 5 ~ y l a s e , trypsinogen and chymotrypsinogen as stated above for the medium. Cr and en%yme released into the medium were expressed as the percentage of the total radioactivity and the total enzyme content, respectively. LDH activity in the medium was expressed as U/mg acinar protein. Protein content was determined by the method of Bradford (18).

RESULTS In the absence of Ca 2+, 6 mM nicotine caused 34% release also

be

of

51Cr

(will

referred to as 51Cr leakage) from freshly isolated pancreatic acini.

When Ca 2+ was added at a concentration of

0.25

mM,

nicotine

induced

only

about 19% release of 51 Cr, which decreased even furthere with increasing concentrations the

of Ca

presence

2+

, attaining a level of

of nicotine

(Fig. IA).

51

also

with

5mM

Ca 2÷

in

The same pattern was observed when LDH

release was evaluated in the absence and should

7% 51Cr-leakage

presence

of

Ca 2÷

(Fig.

IA).

It

be mentioned here that in the absence of both Ca 2÷ and nicotine,

Cr leakage

(basal level) from isolated pancreatic acini

varied

between

10

and 12% (data not shown).

The effect of nicotine on the rate of secretion of and chmotrypsinogen shown in Fig I.B. found

to

be

amylase,

trypsinogen

from isolated acini in the absence and presence of Ca 2÷ is Again, the pattern of secretion of each of the enzymes

the

same

as

that

was

observed for 51Cr leakage and LDH release.

Although the maximal secretion of each of the enzymes by 6 mM nicotine 2+ occurred in the absence of Ca , the rate of secretion of amylase, trypsinogen and chymotrypsinogen the

same.

in the absence or presence of Ca 2÷ was not

found

amylase or chymotrypsinogen

In the next experiment, 51Cr

acini,

in the absence and presence of 1.25 mM Ca 2+, were tested. Fig.2.

and

the effects of nicotine as well as a

agents

in

on

that

of

variety

of

(Fig. IB).

other

shown

be

In the absence as well as in the presence of increasing concentra-

tions of Ca 2+, nicotine caused a greater release of trypsinogen than

are

to

amylase release from freshly isolated pancreatic The

In the presence of Ca 2+, none of the agents

532

results

(nicotine,

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 139, No. 2, 1986

¢1

4O

4°f 35

30

30

~

20

20

~

o~

15

15

~

×

25

Lu

10

#.

a ..i

5

,,, >N Z I,U ,_1

i

i

i

i

i 2

i 3

i 4

i 5

25 20

,,N 0 ~ I-

10

~

5 ~

o

0

Ca 2. (mM) •

2+

Fig. i. Effect_of increasing concentratzons of Ca on the nicotine-induced bl release of (A) Cr and LDH, and (B) trypsinogen (0), chymotrypsinogen ([]]), and amylase (0) from isolated dispersed pancreatic acini. The concentration of nicotine (L-l-methyl-2-[3-pyridyl]pyrolidine, 98-100% free base) was 6 mM. Incubations without Ca z+, contained 0.I mM EGTA. Each value represents the mean + SEM of 6 experiments.

CCK-8,

secretin and mellitin)

snake

venom

produced

any

absence of Ca 2+, however,

with the exception of Ca 2+ ionophore A23187 significant

stimulation in

51

Cr release.

only nicotine and snake venom enhanced

the

and

In the release

of 51Cr from acini.

When amylase secretion was measured, presence

of

Ca 2÷

each

of

the

named in

the

caused

nicotine

to be effective in enhancing the release of amylase.

the absence of Ca 2+, nicotine caused a 225% rise in compared with the corresponding

basal level.

533

absence

amylase

the

a significant

in

found

secretion,

agents

stimulation(125-550%) was

enzyme

above

it was observed that whereas in

of

Ca 2+

secretion,

only In when

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 139, No. 2, 1986

A I - I 1.25 mM Ca 2+

500

Im Without Ca 2+

400

LU

300 200

100

~_

r'T"M=,

~

~

~11

,°°t

600 l

sool-~

-

.:iii

400

I

200 I

100 t.

=k

m~

z_ o

Z

~

>

Z

Fig. 2. Effects of nicotine, peptide secretagogues and membrane active agents ~nn~he release of (A) Cr and (B) amylase from isolated dispersed pancreatic acini in the absence and presence of 1.25 mM Ca 2+. Incubations performed in the absence of Ca 2+ contained 0.I mM EGTA. Each value represents the mean SEM of 6 experiments.

DISCUSSION The finding of a temporal necrosis

and

the hypothesis influx

the

association between the development

of cellular

accumulation of Ca 2+ has led some investigators

that cell death by membrane active toxins

of Ca 2+ into cells (7,10).

However,

is

the significance

mediated

cultures

of

hepatocytes.

All

Sandy

(12)

and

Bellomo

freshly isolated hepatocytes

an

in

pri-

attempts to reproduce this effect in a

freshly isolated hepatocyte system have been unsuccessful and

by

of extracellular

Ca 2+ on toxic cell injury and cell death, could be demonstrated only mary

to support

et at (20) on the

(11, 12, 19).

basis of their studies with

have supported a more significant

534

Smith

role for

early

Vol. 139, No. 2, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

changes in intracellular

Ca 2+ distribution rather than a crucial role for Ca 2+

influx in toxic cell injury. aforementioned

model

Due

systems,

to

the

disparity

of

results

with

we have utilized yet another in vitro system,

that of freshly dispersed rat pancreatic acini to study the role of lular

Ca 2+

in cell injury induced by a variety of agents.

extracel-

One of the advan-

tages of using pancreatic acini is that they retain their inherent high bolic

activity

and

the

meta-

they respond to various endogenous and exogenous factors

that normally affect the e×ocrine pancreas

in vivo.

In the present investiga-

tion,

toxic cell injury was assessed by measuring the release from pancreatic

acini,

of pulse-labeled

cell

damage

51Cr, a method that has been shown to reliably reflect

(21,22).

Our

observation that the pattern of nicotine induced

release of 51Cr and LDH from dispersed acini was the same in the increasing

concentration

of Ca 2+, further corroborates

release as a measure of cell damage.

Furthermore,

presence

of

the validity of 51Cr-

the fact that the

rate

of

release of both 51Cr and LDH by nicotine was more pronounced in the absence of Ca 2+

with a subsequent precipitous

tions

suggests

more susceptible

that

a)

decrease with increasing

Ca 2+

concentra-

the freshly isolated dispersed pancreatic acini are

to nicotine-induced

cell damage in the absence of extracellu-

lar Ca 2+, and b) Ca 2+ plays a protective role against nicotine cell injury.

Similarly, is

also

absence of increased

found

snake venom, which is known to contain a cytolytic to

be

more

extracellular release

of

component,

effective in producing toxic cell injury in the

Ca 2+

than

in

its

presence.

In

addition,

the

51

Cr from dispersed acini induced by nicotine and snake 2+ venom in the absence of Ca is also accompanied by a greater discharge of amylase as compared to that observed in the presence of extracellular Ca 2+ .

In contrast to the findings with nicotine and snake venom, phore

A23187 which creates Ca 2+ channels across the plasma membrane

duced a massive release of lar

the Ca 2+ iono-

Ca 2+

51

Cr and of amylase in the presence of

but not in its absence.

been reported with primary cultures

(23) pro-

extracellu-

Such an observation is similar to what has of

535

adult

rat

hepatocytes

(7).

Thus,

Vol. 139, No. 2, 1986

although

Ca 2+

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ionophore A23187 induced acinar cell injury can be interpreted

as due to entry of Ca 2+ into the cell, neither nicotine nor snake venom cated

acinar

cell

damage

Ca 2+.

In addition, we have also observed that mellitin,

medi-

could be related to the presence of extracellular the

main

cytolytic

component of bee venom (24), which has been shown to reduce viability of hepatocytes in the presence of Ca 2+, induced no damage to creatic

acini

freshly

either in the absence or in the presence of Ca 2+.

fact that mellitin had no

effect

on

51Cr

release,

it

amylase secretion from freshly isolated pancreatic acini. the observation that the acini

isolated

mellitin-induced

amylase

Despite the

greatly

stimulated

This, together with

secretion

from

isolated

was dependent upon extracellular Ca 2+, indicates that mellitin may be a

potential secretagogue for the exocrine pancreas. Thus, although Ca 2+ to

be

appears

necessary for sustained enzyme release induced by secretagogues and by

membrane active agents, a specific role for extracellular toxic

pan-

Ca 2+

in

mediating

cell injury in freshly isolated rat pancreatic acini could not be esta-

blished.

ACKNOWLEDGEMENTS This work was supported by the Medical Research Service of Administration.

the

Veterans

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