changes in cytosolic free calcium concentration in isolated rat parotid cells by cholinergic and...

Vol. 131, No. 3, 1985

September 30, 1985

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 1048-1055

CHANGES IN CYTOSOLIC FREE CALCIUM CONCENTRATION IN ISOLATED RAT PAROTID CELLS BY CHOLINERGIC AND B-ADRENERGIC AGONISTS

Haruo Takemura

Department of Pharmacology, Sapporo Medical College, Sapporo 060, Japan

Received July 29, 1985

The alteration in the concentration of cytosolic free calcium ([Ca2+]i ) in isolated rat parotid cells caused by autonomic agents was directly measured using the Ca-sensitive fluorescent probe, quin2. [Ca2+Ii of unstimulated cells was estimated to be 162.7i3.2 nM in normal medium. Carbachol (CCh) and isoproterenol (ISP) caused a rapid rise in [Ca2+]i in a dose-dependent manner. Maximum increases in [Ca2']i induced by CCh and ISP were approximately 100%

respectively. ~~~1:'" r~~i~'":~~~ 1~~e~~a2t]i,

In Ca-free medium, CCh produced a

resti;g level within 3-4 min, followed by a slow decay and a return to

[Ca2+li. while all doses of ISP tested failed to change

These results suggest that CCh mobilizes Ca2+ from both extracellular and intracellular pools and then results in a rise in [Ca2']i, whereas ISP may slightly mobilize only the extracellular Ca pool. @ 1985 Academic Press, Inc.

A rise in [Ca 2+ Ii is believed to play a key role in the regulation of

amylase release induced by autonomic agents from parotid gland cells (172).

This assumption is based on the indirect evidence reported below (3-9).

First, CCh, a cholinergic agonist, induces amylase release, which is dependent

on extracellular Ca2+ and accompanied by an increased influx of 45Ca2+ into

parotid cells. Second, amylase release stimulated by ISP, a 8-adrenergic

agonist, is inhibited in cells depleted of Ca but not by short-term exposure

to Ca-free medium, although ISP activates the accumulation of cyclic AMP.

Moreover, ISP stimulates 45Ca2+ influx as well as 45Ca2+ efflux. In addition

to these facts, previous studies showed that ISP-induced amylase release is

potentiated by a low dose of CCh (10) but inhibited by high doses of CCh (11).

These stimulatory and inhibitory effects are thought to be dependent on the

elevation of [Ca 2+ 1 i but independent of the accumulation of cyclic AMP.

Abbreviations: BSA, bovine serum albumin; [Ca 2+]i, concentration of cytosolic free calcium; CCh, carbachol; DMSO, dimethyl sulfoxide; EGTA, ethylene bis-(B- aminoethylether)-N,N,N',N'-tetraacetic acid; ISP, (-)-isoproterenol (+)-bi- tartrate

0006-291X/85 $1.50 Copyright 0 I985 by Academic Press, Inc. All rights of reproduction in any form reserved. 1048

Vol. 131, No. 3, 1985 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Despite these facts, no direct measurement of [Ca2+li in parotid cells has yet

been made. The present study was undertaken to measure directly [Ca2t]i using

a Ca selective fluorescent indicator, quin2 (12) and to further investi-

gate the role of [Ca2+li in amylase release.

MATERIALS AND METHODS

KumamZZn2 acetoxymethyl ester (quin2/AM) was obtained from Dojin Chemicals,

BoehringLr; Japan; A23187 from Calbiochem-Behring; BSA (fraction V) from

atropine sulfate from Merck; and CCh, ISP and DL-propranolol HCl from Sigma. Quin2/AM and A23187 were dissolved in DMSO as stock solutions at concentrations of 50mM and lOmM, respectively. The final concentration of DMSO in the incubation medium was 0.1% (V/V),

Isolated parotid cells were prepared from 3 male Wistar rats (220-3003) as described previously (13). The parotid cells obtained were suspended in modified Krebs-Ringer-Hepes (KRH) medium of the following composition (mM) containing 2% BSA: NaCl, 120; KCl, 5.0; CaCl2, 2.0; MgC12, 1.0; Na pyruvate, 5.0; Na glutamate, 5.0; Na fumarate, 2.5; Na S-hydroxybutyrate, 5.0; Hepes, 10.0, 1x107

buffered with KOH to pH 7.4. The cell suspension, containing about cells/ml, was incubated with 50 uM quin2/AM for 45 min at 37'C and

gassed continuously with 02. The cells were then washed twice by centrifuga- tion (50xg,for 3 min) with KRH medium containing 0.2% BSA and maintained at room temperature. Before use, the cell suspension was centrifuged and the cells were resuspended in fresh normal KRH medium or Ca-free KRH medium with 0.1 mM EGTA added, 1-2 x10' cells/ml.

both of which contained 0.2% BSA, at cell concentration of Fluorescence was measured at 37'C in a Hitachi 650-10s

spectrofluorometer equipped with a thermostatically controlled heat exchanger and a magnetic stirrer. The excitation and emission wavelengths were 339nm (5nm slits) and 492nm (10nm slits), respectively. At the end of the analysis, 0.06% Triton X-100 was added to measyy the maximum fluorescence (Fmax) in the presence of an excess amount of Ca (> 1mM) and the minimum fluorescence (Fmin) was measured with 0.5 mM MnC12 (14). Fmax, Fmin and the fluorescence of intracellular quin2 were corrected for changes in cell autofluorescence and for leakage of quin2 in extracellular medium by using 0.1 mM MnC12 (14). A23187 (0.1 uM) did not affect cell autofluorescence and the interference of 0.1 PM A23187 with Fmax was less than 5%. [Ca*+]i was calculated assuming a quin22Ca dissociation constant of 115nM as described by Tsien et al. (12).

The values of the data were given as the mean istandard error. The comparative significance of the values was examined by paired t-test.

RESULTS

The [Ca2+li of unstimulated rat parotid cells in normal medium was calcu-

lated from the fluorescence of quin2 to be 162.7 t 3.2 nM (n=36). The addition

of CCh to the cell suspension caused a rapid increase in [Ca *+I, in a dose-

dependent manner in the range of 0.1 to 100 UM (Fig.1, Table 1). [Ca2+ 1 1

elevated by higher doses of CCh (10, 100 uM) decreased slightly and gradually

for a few min and then maintained its higher level, at least within the

observed time (10 min). Atropine (10 UM) abolished the elevation of [Ca *+li

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O.lyM CCh

1pM ISP

3 350- 1OpM Atropine

5 250- .- 200-

-MM I

‘“‘f

200-

T 150-

.u 150- b&ftJw

10~M CCh 2 min t

100pM CCh

Figure 1. Effects of various doses of CCh and the combination with 1 uM ISP on the quin2 fluorescence in isolated rat parotid cells. Quin2 was loaded as described in METHODS. CCh, ISP and atropine were added at the times indicated by arrows. Calculated Ca2+ (nM) is indicated at the left of each trace.

stimulated by CCh. The maximum rise in [Ca2’li occured at 10 $I CCh and was

about 2 times higher than the unstimulated level.

Table 1. The effect of carbachol and isoproterenol on cytosolic free Ca” in isolated rat parotid cells in normal and Ca-free media

% increase of untreated cell level

Additions Concentration Normal medium Ca-free (0.1 mM CUM) EGTA) medium

: “ , ”

Carbachol 0.1 21.4 ? 4.4 (9) >‘- ;g

1 56.5 t 10.6 (5) :“:”

10 101.4 + 13.8 (9) :% ::

100 94.7 * 10.3 (5)

Isoproterenol 0.1 0.1 2 3.0 (3)

1 5.3 f 2.0' (6)

10 13.3 f 3.3* (4) *:s

100 25.4 + 4.8 (6)

1.4 + 1.4 (3)

9;9 + 3.4* (3) >p :s

13.3 ? 0.4 (3)

*:x 19.9 t2.0 (4)

0 (1)

-2.2 (2)

1.2 (2)

1.2 to.7 (4)

Quin2 was loaded as described in METHODS. The values were expressed as % increase in 1Ca2+Ii after the addition of drugs compared with unstimulated levels. Unstimulated levels of [Ca2+]i in normal medium and in Ca-free medium were 162.7t3.2 nM and 86.8t2.3 nM, respectively. The number of experi- ments is shown in parentheses. “P<O.O5, “;“P < 0.01 vs. unstimulated level.

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1yM ISP 150- t

10!.1M ISP

IOJJM Propranolol

7 z 250-

-m-*M "Ilj-w#qhlkw x- m 150- 22 t t

100~.1M ISP 2 min lOOtiM ISP

Figure 2. Effects of ISP on the quin2 fluorescence in parotid cells. Quin2 was loaded as described in METHODS. ISP and propranolol were added at the times indicated by arrows. Calculated Ca2+ (nM) is indicated at the left of each trace.

The effect of ISP on [Ca2+li indicated b y quin2 fluorescence is shown in

Fig. 2. Although ISP at concentrations of 1 to 100 PM increased [Ca 2+]i dose-

dependently, the degree of increase in [CaZ+li was small (Table 1). ISP

(1 vM), which induced a distinct release of amylase in previous studies

(lO,ll), caused an increase of only 5.3% over resting [Ca *+li. Propranolol

(10 ~JM) inhibited the stimulatory effect of ISP on [Ca *+li.

Previous reports (10,ll) have shown that CCh has both stimulatory and

inhibitory effects on amylase release but not on the accumulation of cyclic

AMP in the presence of 1 UM ISP. To examine the effect of a combination of

CCh and ISP on [Caz+li, 1 UM ISP was added after the addition of various doses

of CCh (Fig. 1). The elevation of [Ca2+ji stimulated by all doses of CCh

tested was unaffected by the addition of ISP.

To determine whether a rise in [Ca 2+li in the presence of CCh or ISP is

dependent on extracellular Ca 2t , the fluorescence of quin2-loaded cells that

were suspended in Ca-free medium containing 0.1 mM EGTA was examined. The un-

stimulated level of [Ca2+li of parotid cells in Ca-free medium was 86.822.3 nM

(n=15). As shown in Fig. 3, the addition of 100 UM CCh to the cell suspension

rapidly increased[Ca2+li, but [Ca2+ji decayed slowly and returned its starting

value within 3 - 4 min. CCh did not change [Ca2+li in the presence of 10 UM

atropine. The increase in [Ca2+li elevated by CCh was dose-dependent in the

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s 5 150-

l@M Atropine lOOtiM CCh

I I 120-

0.1% DMSO O.lpM A23187

O.lyM A23187 2 min 100j~M CCh

Figure 3. Effects of CCh, ISP and A23187 on the quin2 fluorescence in &-free medium containing 0.1 mM EGTA. Quin2 was loaded as described in METHODS. CCh, atropine, ISP, 0.1% DMSO and A23187 were added at the times indicated by arrows. Calculated Ca*+ (nM) is indicated at the left of each trace.

range of 1 to 100 IJM but was only about 20% of the unstimulated level even at

the highest dose (Table 1). On the other hand, 100 nM ISP failed to

increase [Ca 2+ Ii in Ca-free medium, and a transient rise in [Ca 2+li caused by

the subsequent addition of CCh was similar to that in the presence of CCh

alone. Lower doses of ISP (5 10 uM) did not change [Ca2+li (Table 1).

Because of absence of extracellular Ca 2t , CCh-stimulated elevation of

[Ca2+ Ii is ascribed to the release of Ca 2+ from an intracellular Ca store.

To determine the location of this CCh-sensitive store, the alteration in

[Ca2+l 1 caused by CCh in Ca-free medium was compared with that produced by

A23187. A23187 (0.1 uM) increased [Ca2+li more slowly than did CCh, followed

by a slow decay to a new steady value higher than that of untreated cells

(Fig. 3). After [Ca2+ Ii elevated by 100 uM CCh returned to the unstimulated

value, the addition of A23187 to the cell suspension produced a comparative

increase in [Ca2+li in the p resence of A23187 alone.

DISCUSSION

The [Ca2+li of unstimulated rat parotid cells in normal medium containing

2 mM Ca2+ was about 160 nM. This value is very close to that reported for

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[ ca2+ ] which was measured using quin2 in exocrine pancreatic acini (15,16). 1

On the other hand, the removal of Ca 2t from suspension medium and the addition

of 0.1 mM EGTA led to a value which was half that of [Ca 2+ Ii in normal medium.

Previous investigations (10,ll) showed that CCh induces amylase release

in the range of 0.1 to 100 UM and the maximum release, which is about two

times that of control, occurs at a concentration of 10 IJM. This dose-depend-

ent relationship corresponded to that of [Ca2'li response to this agonist

(Fig. 1, Table 1). However, all doses of CCh failed to stimulate amylase

release in Ca-free medium (lO,ll), although CCh caused a transient and slight

increase in [CaZtli (Fig. 3). In the absence of extracellular Ca2+, CCh

stimulated the increase in [Ca ?+li in pancreatic acini (15) more than in

parotid cells (Table 1) and was still able to elicit amylase release in the

former (17). Therefore, the elevation (>160 nM) of [Ca2+li above resting

level in the presence of extracellular Ca 2t is presumed to result in the

induction of amylase release from parotid cells. A transient rise in [CaZtli

stimulated by CCh in Ca-free medium seems to be associated with the early

phase of potassium movement (2) rather than amylase release. Such a CCh-

sensitive Ca pool is thought to possibly be located in plasma membrane. This

supposition is based on the following observations. First, A23187, which

releases Ca2+ from liver mitochondria (18) and from pancreatic microsomes

(19), increased [Ca2'li more slowly than did CCh, and was then followed by a

slow decay to a new steady level. A similar increase in (Ca2t]i, even after

the addition of CCh, also occured (Fig. 3). Second, it is suggested that

methacholine, a cholinergic agonist, provokes a breakdown of phosphatidyl-

inositol 4,5-bisphosphate which is involved in Ca 2t release from the plasma

membrane in parotid acinar cells (20). In addition, inositol 1,4,5-trisphos-

phate, which is a product of this breakdown, releases Ca 2+ from platelet

membranes (21). Third, there is evidence from studies of 45 Ca fluxes showing

that a CCh-sensitive Ca pool is located in or near the plasma membrane (22).

Thus, it seems likely that CCh releases Ca 2t from an intracellular store

(presumably plasma membrane), accompanying a continuous increase in Ca 2t entry

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from the extracellular pool, and then results in a rise in [Ca*+] i, which

induces amylase release.

Studies of 45Ca2+ efflux support the hypothesis that 8-adrenergic

agonists utilize intracellular pool(s) of Ca 2+ to promote amylase release (2).

However, the present observations showed that ISP failed to elevate [Ca 2t Ii in

Ca-free medium, despite the fact that ISP increased [Ca *+li slightly, being

dependent on extracellular Ca 2+ . The discrepancy between the increase in 45Ca

efflux and no change of [Ca*+]. 1 may be explained as follows. Wallach and

Schramm (23) have reported that the secretary granules have the highest Ca

content among the subcellular fractions and most of the Ca is secreted con-

comitantly with the exportable protein. The amount of amylase release induced

by 1 uM ISP in Ca-free medium was similar to that in normal medium (10,ll).

It is, therefore, probable that amylase and 45ca2t are released by exocytosis

without altering [Ca2+li, and that the contribution of Ca2+ to ISP-induced

amylase release is small.

Both stimulatory and inhibitory effects of CCh on 1 uM ISP-induced

amylase release have been supposed to be ascribed to the degree of elevation

of Ca*+ rather than that of cyclic AMP accumulation (10,ll). The direct

2+ measurement of [Ca Ii using quin2 seems to confirm this supposition. Since

ISP did not modify ICaztli in the presence of CCh, it appears that [Ca*'l 1

might regulate amylase release induced by cyclic AMP as suggested by Butcher

and Putney (2). The inhibition of ISP-induced amylase release occurred at

concentrations above 1 I-IM CCh (11). Therefore, the increase of more than 50%

in [Ca 2t 1, may possibly bring about the inhibition of amylase release induced

by ISP. In addition, it provides further support for the previous assumption

that a small alteration in [Ca2+li subtly regulates amylase release when the

accumulation of cyclic AMP is not modified (13).

ACKNOWLEDGMENT : The author wishes to thank Professor H. Ohshika for advice and encouragement.

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REFERENCES

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215-249. 3. Kanagasuntheram, P. and Randle, P.J. (1976) Biochem. J. 160, 547-564. 4. Leslie, B.A., Putney, J.W., Jr., and Sherman, J.M. (1976) J. Physiol. 260,

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Physiol. 236, E745-E762. 18. Reed, P.W. and Lardy, H.A. (1972) J. Biol. Chem. 247, 6970-6977. 19. Wakasugi, H., Kimura, T., Haase, W., Kribben, A., Kaufmann, P. and Schulz,

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555-560. 21. Adunyah, S.E. and Dean, W.L. (1985) Biochem. Biophys. Res. Comm. 128,

1274-1280. 22. Poggioli, J. and Putney, J.W., Jr, (1982) Pflugers Arch. 392, 239-243. 23. Wallach, D. and Schramm, M. (1971) Eur. J. Biochem. 21, 433-437.

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changes in cytosolic free calcium concentration in isolated rat parotid cells by cholinergic and...

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