278

Bioc:limicaet BiophysicaActa, 1134(1992)278-284 © 1992ElsevierSciencePublishe,.~B.V. All rightsreserved0167-4889/92/$05.00

BBAMCR 13124

Relationship between cytosolic Ca 2+ concentration and amylase

release in rat parotid acinar cells following muscarinic stimulation Yosuke Tojyo, Satoko Matsui, Akihiko Tanimura and Yoshito Matsumoto Department of Dental Pharmacology, School of Dentistry, Higashi Nippon Gakuen Unicersity, Hokkaido (Japan)

(Received4 September 1991)

Keywords: CytosolicCa2+ concentration;Amylaserelease; Muscarinicstimulation;Rat parotid acinar cell Carbachol (CCh), a muscarinic-cholinergic agonist, increased both cy.tosolic free calcium concentration ([Ca2+]i) and amylase release in rat parotid acinar cells or acini in a dose-dependent manuer. Treatment of acinar cells with the intracellular Ca2+ antagonist, 8-(N,N-diethylamino)oetyl-3,4,5-trimetboxybenzoate (TMB-8), or the intracellular Ca 2+ chelator, 1,2-bis(Oaminophenoxy)ethane-N,N,N',N'-tetraaeetic acid (BAPTA), strongly attenuated the increases in [Ca2+]i evoked by CCh, bat amylase release from acini was not significantly suppressed by the treatment with TMB-8 or BAPTA. Low concentrations (0.02-0.5 p.M) of ionomycin, a Ca2+ ionophore, caused increases in [Ca2+]i comparable to those induced by CCh, but the same concentrations had only a little effect on amylase release. The protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate TPA), stimulated amylase release in quantities similar to those induced by CCh, although TPA alone did not cause any change [Ca2+]i. Combined addition of TPA and ionomycin potentiated only modestly amylase release stimulated by TPA alone. Staurosporine, a protein kinase C-inhibitor, similarly inhibited both the CCh- and TPA-induced amylase release. These results suggest that an increase in [Ca2+]i elicited by CCh does not play an essential role for inducing amylase release in rat pamtid acini. Amylase release by muscadnic stimulation may be mediated mainly by activation of protein kinase C.

I

Introduction Amylase exocytosis from rat parotid acinar cells is induced by activating either of two distinct signaling mechanisms. One involves an accumulation of adenosine 3',5'-cyclic monophosphate (cAMP) via activation of/3-adrenergic receptors, resulting in a large amount of amylase release (see Ref. 1 for review). ']'his secretory mechanism appears to be independent of changes in the cytosolie Ca 2+ concentration ([Ca2+]i) [2,3]. Another mechanism for amylase release is activated by stimulation of muscarinic-cholinergic, a-adrenergic, or substance P receptors, leading to phosphoinositide

Abbreviations: CCh, carbachol; [Ca2+ ]t, cytosolicfree calcium concentration; TMB-8, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxy. benzoate; BAPTA, 1,2-bis(O-aminopheno~)ethane.N,N,N',N'.te. traacetic acid; TPA, 12-O-tetradeeanoylphorbol-13.acetate;cAMP, adenosine3',5'-cyclicmonophosphate. Correspondence: y. Tojyo, Department of Dental Pharmacology, School of Dentist~, Higashi Nippon Gakuen University,IshikariTobetsu, Hokkaido061.02,Japan.

breakdown which yields inositol 1,4,5-trisphosphate (IP 3) and diacylglycerol (see Refs. 4, 5 for review). Stimulation of these three receptors evokes relatively limited amylase release together with fluid secretion and ion transport. Previous studies using fluorescent Ca 2+ indicators such as quin2 and fura-2 have shown that stimulation of muscarinic receptors causes a large increase in [Ca2+]i in parotid acinar cells [6-8]. The [Ca2+]t response is believed to be attributed to the IP3-induced Ca 2+ release from intracellular stores and the entry of Ca 2+ across the plasma membrane. Since removal of Ca 2+ from extracellular medium reduces or abolishes amylase release induced by muscarinic agonists [9-11], the rise in [Ca2+]i has been assumed to play a crucial role in stimulating the amylase release. However, the precise role of [Ca2+]i is still not fully established. To assess whether amylase release from rat parotid acini by muscarinic stimulation is primarily mediated by an increase in [Ca2+]i, we examined the relationship between changes in [Ca2+]i and amylase release. The results suggest that the increase in [Ca2+]t is not essential to induce amylase release.

?79 Materials and Metl:~ls

Materials Carbachol (CCI',), hyaluronidase (type l-S), trypsin (type Ill), trypsin inhibitor (type II-S), bovine serum albumin (BSA), collagenase (type II), 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), ionomycin and stat~rosporine were purchased from Sigma (St. Louis, MO, USA). Hanks' balanced salt solution was from Gihco (Chagrin Falls, OH, USA) and Nissui Pharmaceutical (Tokyo, Japan). Fura-2 acetoxymetbyl ester (fura-2/AM), the acetoxymethyl ester of 1,2-bis( O.aminophenoxy)ethane.N,N,N ,,N '-tetraacetic acid (BAPTA/AM), EGTA, Hepes were from Dojin Laboratories (Kumamoto, Japan). All other reagents were from Wako Pure Chemical (Osaka, Japan).

Preparation of parotid acini and dissociated acinar cells Male Wistar-strain rats were anesthetized with diethyl ether and killed by cardiac puncture. Dispersed acini were prepared as previously described [12]. Briefly, parotid glands were minced finely and incubated for 60 min at 37°C in Hanks' balanced salt solution buffered with 20 mM Hepes-NaOH (pH 7.4) (HBSS-H), containing collagenase (0.8 mg/ml) and hyaluronidase (0.25 mg/ml). After dispersion, the acini were filtered through nylon mesh, washed twice with HBSS.H containing 0.1% BSA, and then suspended in fresh medium. The isolated acini had well-preserved acinar structures and maintained the ability to respond to stimuli for amylase release. Dissociated acinar cells were prepared by the method of Merritt and Rink [7] with our modifications [3,12]. Briefly, minced glands were incubated for 10 rain at 3"PC in HBSS-H containing trypsin (0.5 mg/ml) and 0.1% BSA, washed with a Ca2+-free medium containing 2 mM EGTA, and then incubated for 5 min with trypsin inhibitor (0.5 mg/ml). After washing, tissue was incubated for 20 rain in HBSS-H containing collagenase (0.33 mg/ml) and 0.1% BSA. Dispersed cells ~ere filtered through nylon mesh, washed twice, and resuspended in fresh HBSS-H containing 0.1% BSA. This cell suspension consisted of single cells and double- or triple-cell masses.

Assay of amylase release A 1.0 ml portion of acini suspension was transferred into a glass tube and stimulated with secretagogues for 30 rain at 37°C. After incubation, the medium was collected by filtration through filter paper. To measure the total amylase content in the acini, a portion of the suspension was homogenized with a Polytron homogenizer. Amylase activity in media and homogenates was measured by the method of Bernfeld [13]. The released amylase was expressed as percent of the total amylase in the acini.

Assay of leakage of lactate dehydrogenase (LDH) The LDH leakage was assayed by a protocol similar to that tor amylase release. The LDH activity was measured by the method of Wr6blewski and La Due [14].

Measurement of/Ca 2+L Dissociated acinar ceils were incubated with 2 /~M fura-2/AM for 45-60 rain at 37°C. The fura-2-1oaded cells were washed twice and resuspended in fresh HBSS-H containing 0.1% BSA. In experiments to assess the effects of the intracellular Ca2÷ chelator BAPTA on [Ca2+]i, cells were incubated for 30 rain with BAPTA/AM after loading with fura-2. The control ceils were incubated for 30 rain in the absence of BAPTA/AM. The fluorescence of fura-2-1oaded cells was measured in a Hitachi F-2000 spectrnfluorimeter (Hitachi, Tokyo, Japan) with excitation at 340 and 3 ~ nm and emission at 510 rim. The cuvette was thermostatically controlled at 3"PC, and the cell suspension was continuously stirred. [Ca2+]i was calculated from the ratio of fluorescence, as described by Grynkiewicz et al. [15]. Maximum fluorescence was determined by lysing the cells with 0.1% Triton X-100, and minimum fluorescence was determined by the addition of 30 mM Tris base and 5 mM EGTA.

Results l~ffect of CCh on [Ca z +]i and amylase release The [Ca2+]i of unstimulated parotid acinar cells was estimated to be 87.2 + 3.0 nM (mean + S.E., n = 18). Stimulation with various concentrations (0.1-100/zM) of CCh increased [Ca2+]i in a dose-dependent manner. Fig. 1A shows a typical change in [Ca2+]i evoked by 10 /~M CCh. The increase in [Ca2+]i reached a peak value within 10 s after the addition of CCh, then gradually declined to a sustained level, but did not return to the resting level during stimulation. CCh also stimulated amylase release from parotid acini in a dose-dependent manner. Fig. 2 shows concentration-response relationships of the elevation in [Ca2+]i and amylase release induced by CCh. The dose-response curve for the CCh-induced amylase release was closely parallel to that for the peak [Ca2+]i, while there was no correlation between the sustained [Ca2+]i and amylase release.

Effects of TMB-8 and BAPTA on an increase in [Ca z +]i and amylase release stimulated by CCh To assess whether CCh-induced amylase release is directly dependent on the peak [Ca2+]i, the effects of TMB-8, which is thought to inh~it Ca2+ mobilization from intracellular stores [16], were examined. When TMB-8 ( ~ 10 ~tM) was added before stimulation with CCh, the peak [Ca2+]i elevated by 10/~M CCh signifi-

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Fig. 3. Effect of TMB-8 on amylase release and 'he increase in [Ca2+ ]i induced by CCh. CCh was added 5 min after the addition of TMB-8. For amylase release, parotid ~ci,fi were stimulated for 30 rain with 10 /tM CCh. The released amylase was expressed as percent of total amylase of the acini. ( o o), CCh-stimulated amylase release; ( o . . . . . . O), basal release. For measurement of lCa2+ ]i, fura-2-1oaded acinar cells were stimulated with 10/tM CCh in the presence of various concentrations of TMB-8, and the peak

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2~o 4~0 n~o Time (seconds) Fig. l. (A) A typcialchange in [Ca2÷li inducedby 10 pM carbachol (CC). CCh was added to cell suspensionat the time indicated by the arrow. (13)Effectof TMB-8on CCh-inducedincreasein [Caz+ l,. TMB-8 (30/tM) was added 5 min before stimulationwith 10/tM CCh. (C) Effect of EAPTA on CCh-inducedincrease in [Ca2+]iA~nar cellswere loaded with BAPTAby pro-incubatingthem for 30 min with 10 F.M EAPTA/AM. The cells were then washed and stimulatedwith 10 IzM CCh.

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[Ca2+] I. For amylase release, parotid acini were stimulated for 30 rain with carbachol (CCh). The released amylase ~vas expressed as percent of total amylase of the acini. Valnes are means:l:S.E, of eight experiments. For measurement of [Ca2+ ]l, fura-2-1oaded acinar cells were stimulated with CCh and the peak (o e) and sustained (e..... -4)) levels of [Ca2+ li were measured. The sustained

[Ca2+lI was monitored 5 min after addition of CCh. Values are means+ S.E. of three experiments.

cantly decreased with increasing concentrations of TMB-8 (Fig. 1B). Fig. 3 shows the relationship between the peak [Ca2+]i and amylase release in the presence of various concentrations of TMB-8. The ICso value (the concentration which reduces the peak [Ca2+li by 50%) for TMB-8 was about 14 ~ M , and 50/~M TMB-8 suppressed the CCh-induced rise in [Ca2+]i to 17% of that observed in the control cells. Despite the noticeable attenuation of the rise in [Ca2÷]i, amylase release stimulated by 10 ~ M CCh was not significantly inhibited by TMB-8, although the basal level of amylase release increased somewhat at 30 and 50/~M TMB.8 (Fig. 3). As the intraceilular Ca chelator BAPTA is believed to buffer changes in [Ca2+] i [17], we tested the effects of BAPTA on increases in [Ca2+] i and amylase release in response to CCh. For loading with BAPTA, parotid acini or acinar cells were pro-incubated for 30 min with 5 and 10 ~ M B A P T A / A M . In the control cells pro-incubated without B A P T A / A M , stimulation with 10/zM CCh elevated [Ca2+] t from a resting level of 127.5 + 3.5 nM to a peak value of 367.7 :t: 44.2 nM (means :t: S.E., n = 3) within 10 s. l_~ading the cells with BAPTA markedly attenuated the peak [Ca2+] i induced by CCh, although a slow and sustained increase i n [Ca2+]i was observed (Fig. 1C). The plateau [Ca2+] i measured at 5 min after addition of CCh was 184.0 + 8.9 and 169.5:1: 9.7 nM in cells pro-treated with 5 and 10 /~M B A P T A / A M , respectively. In contrast, the loading with BAPTA had only a little effect on amy|age release. from parotid acini in response to 10/~M CCh (Table I).

281 TABLE I

TABLE II

Effect of BAPTA on carbachol.induced amylase release

Effect of ionomycin on lCa z * J~ and amylase and LDH release

Parotid acini were pre-incubated for 30 rain with or without BAFTAAM. After washing, the acini were stimulated for 30 rnin with l0/.tM carbachol (CCh). The released amylase was expressed as percent of total amylase in the acini. Values are means+S.E, of five experiments.

For measurement of [Ca2+ it, fura-2-1ueded acinar cells were stimulated with various concentrations of ionm~cin, and the ba~l and peak levels of [Ca2*]i were measured, The values in parenthesns show the basal levels in each Iffoup. Values are means+ S.E. of three experimant~. * p < 0.05, as compared with the basal value (paired t-tes!). Fo~ amylase and lactate delwdrogenase (LDH) release,pamtkl acmt were incubated for 30 rain with or without iouonv~n. The release was t:xpressed as percent of the total cellular content. Values are means+S.E, of four experiments. * P