309

Biochimica et Biophysica A cta, 583 (1979) 309--319 © Elsevier/North-Holland Biomedical Press

BBA 28841

AMYLASE RELEASE FROM RAT PAROTID GLANDS II. CALCIUM KINETICS

J.C. K U S E K

Department of Pharmacology, Universityof lUinois,College of Medicine, Chicago, IL (U.S.A.) (Received August 29th, 1978)

Key words: Amylase release; Stimulus-secretion coupling; Ca2+;Energy dependence; Cyclic AMP; (Parotid gland)

Summary The kinetics of 4SCa2+ uptake, efflux, and calcium potentiation of amylase release by slices of rat parotid glands were examined. Pretreatment of the tissue with 11.25 mM 4SCa2÷ medium increased the total tissue 4Scalcium content. Lanthanum (1 mM) decreased tissue uptake, blocked the slow components of exchange and appeared to inhibit transcellular calcium movement. Neither dibutyryl cyclic AMP nor caffeine caused consistently significant effects on 4SCa2÷ kinetics, or total 4Scalcium content. Carbamylcholine increased the initial rate of 4SCa2+ uptake, but had no effect on total uptake. Elevation of the extracellular Ca2÷ concentration to 11.25 mM during stimulation of amylase release resulted in an initial decrease in the rate of amylase release followed by a potentiation of release which developed slowly, requiring 40--50 min to reach the maximal response. The inability to detect release-related changes in either calcium influx or mobilization, and the lengthy times and high Ca2÷ concentrations required to achieve calcium potentiation suggests that calcium does not couple amylase release.

Introduction

Calcium is an essential component of stimulus-secretion coupling in a wide variety of tissues [ 1 ]. In many tissues, as exemplified by the adrenal medulla, the entry of extracellular calcium into the secretory cells promotes the release of the granular contents; and a reduction of the extracellular Ca2÷ concentra-

310

tion causes a p r o m p t reduction in release [ 2,3 ]. The release of amylase from rat parotid glands appears to be influenced by calcium. Pre-incubation of glandular tissue with a calcium-chelating agent reduces dibutyryl cyclic AMP-stimulated amylase release [4], whereas pre-incubation in media with elevated calcium concentrations potentiates the secretory response to a variety of stimulants [5]. These effects require long-term pretreatment of the tissue. The short-term responses to stimulants of amylase release are unaffected by changes in the extracellular Ca 2÷ concentration [6-9]. Therefore, the regulatory influence of calcium on amylase release is probably not due to a stimulus-induced influx of Ca 2÷ across the plasma membrane, and the calcium potentiation of release is probably related to other mechanisms. In this study, the role of calcium in amylase release was reinvestigated. Uptake of calcium into the functional pool associated with release was determined by following the time course of development of calcium-potentiated responses. Uptake and efflux of isotopic calcium were examined in order to determine if release is associated with an alteration in membrane permeability to calcium, a change in the total calcium content of the tissue, or a mobilization o f intracellular calcium. Methods A batch incubation m e t h o d was used to determine 4SCa2* uptake. Rat parotid gland slices were prepared as previously described [5]. The slices from the glands of 3--6 animals were pooled and equilibrated in 300--600 ml of control medium for 60 min. Slices were washed and randomly distributed into parafilm-covered 100-ml beakers containing 1.25 mM Ca 2÷ medium with or without stimulants of release. The ratio of tissue to medium was approx. 1 mg/ml. Following a 30 min equilibration in the experimental medium, 45Ca2÷ uptake was initiated b y addition of isotope in a solution containing sufficient Ca 2÷ to either maintain 1.25 mM Ca 2÷ or to increase the concentration to 11.25 mM Ca 2÷ (final specific activity of medium = 0.36 Ci/mol Ca2÷). Tissue samples (approx. 10 mg wet weight) were removed at timed intervals, rapidly washed with 10 ml of nonradioactive medium in a small Buchner funnel under mild vacuum, lightly blotted, and weighed. Tissue samples were digested overnight at 40°C in 0.8 ml of Nuclear Chicago Scintillator ~ tissue solubilizer, neutralized with glacial acetic acid, and 10 ml of scintillation cocktail was added. The extracellular space was determined b y parallel [3H]inulin uptake measurements for each preparation (specific activity of medium = 2.5 pCi/ml). Total tissue uptake of calcium was calculated from the following equation: nmol Ca :+ _ nmol Ca 2+ × r( 4SCa dpm/mg tissue 1 -- ( 3 H d p m / m g tissue t7 mg tissue pl medium L\4-TCaa-dp-m~med-~mum.lt3H dpm/pl m e d i u m / J The superfusion m e t h o d was used to determine 4SCa:+ efflux. Tissue samples (40--50 mg wet weight) were placed in tissue holders and equilibrated with control medium by superfusion (approx. 1 ml/min) for 60 rain. Since slight variations in efflux curves were observed between preparations, t w o successive washouts were performed on each tissue sample. Samples were superfused with

311 4SCa2+ containing medium (0.36 or 0.18 Ci/mol Ca 2+) for 60 min in order to load the calcium pools. Washout was accomplished b y a 40 min superfusion with nonradioactive medium. The initial washout (control) was followed b y a second 60 min loading superfusion and a 40 or 45 rain washout with experimental medium. Effluent from the washouts was collected for l-rain intervals and 0.5-ml samples were added to 10 ml of scintillation cocktail. The cocktail consisted of: xylene/p-dioxane/methylcellosolve ( 1 : 3 : 3 , v/v), 5% (w/v) naphthalene, 1% PPO, and 0.1% POPOP. The radioactivity was measured in a Packard Tri-carb liquid scintillation spectrometer, and efficiency was determined b y internal standards. Efflux was analyzed using the following equation for multiple exponential decrease: Y = Y~ e-k~t + Y2 e-k2t + Y3 e -k3t + ... Y~ e is the base of the natural logarithms, k is the rate constant, and t is the time (min) from the initiation of the washout. Experimental data were fitted to this model b y the m e t h o d of Riggs [10]. Most efflux curves appeared to follow triexponential functions; the c o m p o n e n t with the slowest turnover was designated as phase III and the fastest c o m p o n e n t as phase I. The rates of efflux of the three phases are expressed as half-times (tl/2) which are equal to 0.693/k. The superfusion m e t h o d was also used to determine the rate o f change o f amylase release in response to elevation of the external Ca 2+ concentration during submaximal stimulation of release. Tissues were equilibrated with control medium (1.25 mM Ca 2+) b y superfusion for 60 min. Following equilibration, all samples were exposed to submaximal concentrations of a secretory stimulant and re-equilibrated for 40 min. Half of the samples were then switched to 11.25 mM Ca 2÷ medium with stimulant and the remaining samples served as controls. Amylase release was monitored for an additional 90--120 min. The amylase activities of the effluent and tissues were determined b y a saccharogenic m e t h o d and the secretory response is expressed as the percent of the original tissue activity released per min. The differences in the rates of release between the controls (1.25 mM Ca 2÷) and experimentals (11.25 mM Ca 2÷) were taken as a measure of the time course of functional loading of the calcium sites associated with amylase release. Dibutyryl cyclic AMP, caffeine, and carbamylcholine were obtained from Sigma Chemical Co. (St. Louis, MO). Isotopes and Nuclear Chicago Scintillator solubilizer were purchased from Amersham/Searle Corp. (Arlington Heaghts, IL). The specific activity of [3H]inulin was 300 Ci/mol. 4SCaC12 had an activity of 1.0--1.4 mCi/ml. The Ca 2÷ concentration of the solution was adjusted to meet the needs of the experiment and the final specific activity was 0.1--0.4 Ci/mmol Ca 2÷. Results

Calcium uptake In initial experiments, the uptake of 4SCa2÷ was monitored for 80 min (Fig. 1). The size of the calcium space, corrected for the inulin space, did n o t increase significantly after 40 rain of incubation (40 rain 4SCa2+ space = 1.441 + 0.108/A/mg tissue; 80 min 4SCa2+ space = 1.440 + 0.064 pl/mg; mean +S.D., n =

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4). The calcium uptake from 11.25 mM Ca2÷ medium (4.535 -+ 0.426 nmol/mg; n = 3) was considerably greater than that from control medium containing 1.25 mM Ca2÷ (1.762 -+ 0.140 nmol/mg), and followed an irregular pattern which was not consistent from preparation to preparation. However, the uptake had stabilized by about 40 rain. In subsequent experiments the 80-rain time points were omitted. Pretreatment with 1 mM La 3÷ markedly altered the time course and decreased the amount of 4SCa2÷ accumulation. Uptake in La3÷-treated tissues paralleled the time course of equilibration of the extracellular space with [3HIinulin (Fig. 2). Lanthanum reduced accumulation by 56%. Total calcium uptake (40 min incubation corrected for inulin space) of control tissues was 1.791 _+0.173 nmol Ca2+/mg tissue (mean _+S.D., n = 4); uptake by La3+-treated tissues was 0.795 _+ 0.134 nmol Ca2÷/mg tissue. Carbamylcholine caused an increase in 4SCa2÷ uptake at early time points. However, it had no significant effect on the total tissue uptake at 40 min (Fig. 3). Caffeine (30 mM) had no effect on either the initial rates or the final extent of 4SCa2÷ uptake. The initial uptake (1--10 rain) during exposure to 1 mM dibuo

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tyryl cyclic AMP tended to be less than control, but the effect was not statistically significant in every preparation. Total tissue uptake was unaffected by dibutyryl cyclic AMP (results not shown).

Calcium efflux Typical washout curves could be resolved into three kinetic phases. The most rapid component, phase I, was observable during the first 6--8 min of washout;

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the intermediate component, phase II, was most prominent during the period between the 7th and 24th min of perfusion; and the slowest component, phase III, occupied the last 15 min of the washout. Variations in the kinetic constants of the three phases were observable between preparations and therefore two consecutive washouts were performed on each preparation. A slight elevation of the second washout was always observed, but the half-times of washout of the three phases were similar. Equilibration o f the tissue for 60 min with 11.25 mM 4SCa2÷ medium produced a substantial elevation of the washout curves (Fig. 4) with increases in the size of the three kinetic phases. Washout with La3+-containing medium markedly altered 45Ca2÷ efflux (Fig. 5). During the first 30--36 min of washout the rates of efflux appeared to be increased, but therefore, a new kinetic phase (phase III2) with an extremely low rate of turnover was observed. The size and half-time of phase III2 could n o t be determined with any degree of precision even when the washout was extended to 55 min, and thus, efflux during La 3÷ exposure was n o t resolved into its kinetic components. Caffeine (30 mM) and dibutyryl cyclic AMP (1 raM) had no appreciable effect on 4SCa2+ efflux (results n o t shown).

Functional loading o f the critical calcium sites Alterations in the Ca2÷ concentration of the medium had no significant

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effect on spontaneous (unstimulated) amylase release, and thus, the sensitivity of secretion to changes in extracellular Ca2÷ and the time course of developm e n t of calcium potentiation was examined during stimulated release. Amylase release was initiated by exposing tissue samples to secretagogues it, control medium (1.25 mM Ca2÷). Following a 40 min exposure to stimulants, half of the samples were switched to stimulant-containing media with altered Ca 2÷ concentrations, and the differences in the rates of release between the two sets of samples were monitored for 90--120 min. Elevation of the Ca 2÷ concentration from 1.0 to 3.0 mM failed to produce consistently significant increases in stimulated release and 10.0 or 11.25 mM Ca 2+ was required before calcium-potentiation was readily demonstrable. A significant decrease in amylase release was usually observed during the first measurement (10 min) after the change to 11.25 mM Ca:+ medium. Thereafter, Ca2+ potentiated the responses to reduction of H ÷, caffeine, and dibutyryl cyclic AMP (Fig. 6). The calcium potentiation appeared to reach a m a x i m u m 40--50 rain after elevation of the extracellular Ca:÷ concentration, and remained stable for the remainder of the experiment. Discussion

The ability of calcium to potentiate the release of amylase was reinvestigated by studying the response to changes in extracellular Ca 2÷ during stimulation of

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secretion. The initial response to 11.25 mM Ca2÷ was a transient decrease in the rate of release. This effect is likely to be due to the direct 'stabilizing' action of extracellular Ca2÷ on excitable membranes [11,12]. Calcium potentiation of stimulated release required substantial elevation of the external Ca 2÷ concentration for prolonged periods. This lack of sensitivity to calcium suggests that either the release-related pool of calcium is very stable and n o t easily altered, or that calcium does not function as the rate-limiting factor in release. Elevation of the external Ca 2÷ concentration to 1 1 . 2 5 m M resulted in increases in total tissue 4SCa2÷. However, the time course of uptake was irregular and did not correspond to the time course of development of calcium potentiation. An early c o m p o n e n t o f uptake which probably represents extracellular binding was prominent and would likely mask secretion-related events. The uptake of 4SCa2÷ from 1.25 mM Ca2* medium followed a regular and consistent pattern which allowed investigation of the effects of various secretagogues. Carbamylcholine, an agent which stimulates K ÷ efflux in the presence of extracellular Ca 2÷ [9,13], increased 4SCa2* uptake at early time points w i t h o u t

318

affecting the total 4sCa2’ content. These results suggest an increase in the turnover of tissue calcium possibly due to an increase in membrane permeability. Koelz et al. [14] have reported carbamylcholine-induced increases in both the rate and extent of 4sCa2+ uptake by isolated parotid cells. The difference in results may relate to differences in experimental conditions. Lanthanum appeared to inhibit transmembrane calcium flux as evidenced by limitation of 4sCa2+ uptake to the extracellular space and by blockade of a component of phase III washout. La3+ also stimulated the earlier phases of washout, presumably by competing with Ca*+ for binding to external sites. It has been postulated that competition and displacement of external Ca*’ is responsible for La3+-induced stimulation of spontaneous release from the adrenal medulla and the neuromuscular junction, and that inhibition of Ca*’ influx blocks stimulated release in these tissues [15,16]. In the parotid gland, La3+ enhanced both spontaneous and stimulated release, and pretreatment of the tissue in 11.25 mM Ca*+ medium potentiated the effects of La3+ [5]. In contrast, 30 min pre-incubation of the neuromuscular junction in 10 mM Ca*+ reduces La3+ stimulation of spontaneous release [ 171. Thus, although the effects of La3’ on parotid calcium are similar to those in other tissues, the characteristics of its effects on release are different. In view of the fact that La3+ also produces effects distinct from its Ca*’ -antagonist activity, e.g. inhibition of (Na’ + K’)-ATPase [18], and alterations in membrane fluidity [ 191, the assignment of a mechanism to the stimulator-y effect of La3’ is premature. Neither dibutyryl cyclic AMP not caffeine produced consistent effects on the initial rate or total uptake of 4sCa2+. Thus, amylase release is not accompanied by an influx of Ca*+, and this result is consistent with the observation of other investigators that reduction of the extracellular Ca*’ concentration has no immediate effect on amylase release. Another possible mechanism for the involvement of calcium is intracellular redistribution, i.e. mobilization of a sequestered store of calcium into the cytoplasm and redistribution to sites associated with secretion. However, the kinetics of 4sCa2+ washout during either caffeine or dibutyryl cyclic AMPinduced release were unchanged. Thus a sustained mobilization of intracellular calcium appears unlikely. Recently, Kanagasuntheram and Randle [20] reported that dibutyryl cyclic AMP or isoproterenol induced a transient release of 4sCa2+during perfusion of an isolated parotid cell preparation with Ca*+-free medium. However, 4sCa2+ efflux quickly returned to control values while amylase release continued at elevated rates. The design of the present experiments precludes the detection of transient changes during the first minute or two of washout since the stimulants of release were introduced simultaneous with the change to nonradioactive medium. However, if a mobilized pool of calcium were coupling release, one would expect to observe evidence of mobilization during development and maintenance of stimulated release. It appears that neither the influx of extracellular Ca*’ nor the mobilization of intracellular Ca*’ is associated with amylase release. Yet, calcium plays some role in release as evidenced by calcium potentiation of stimulated release. These two results are not consistent with classical models of stimulus-secretion coupling involving some form of calcium movement. However, if a pool of

319 parotid calcium were always present at some critical site associated with release, the need for trans- or intracellular calcium movement would be eliminated. In such a 'static-calcium' model, calcium would not act as the coupler which turns release on and o f f in response to a stimuli; although, the amount of calcium present at the critical site could influence the magnitude of the secretory response. In a companion communication [5] it was concluded that cyclic AMP was not an obligatory component o f release. Thus, it appears that while both cyclic AMP and calcium are involved in amylase release, neither is a coupler c o m m o n to all stimulants of release. Acknowledgement

This investigation was supported by National Institutes of Health grant DE 04081. References 1 Rubin, R.P. (1970) Pharmacol. Rev. 22, 389---428 2 Douglas, W.W. (1968) Br. J. Pharmacol. 34, 4 5 1 - - 4 7 4 3 Douglas, W.W. (1975) In H a n d b o o k of Physiology, Sect. 7: Endocrinology, Vol. 6: Adrenal Gland, (Greep, R.O. and Astwood, E.B., eds.), pp. 367--388, American Physiology Society, Washington, D.C. 4 Selinger, Z. and Naim, E. (1970) Biochim. Biophys. Acta 203, 335--337 5 Kusek, J.C. (1979) Biochim. Biophys. Acta 583, 295--308 6 Bdolah, A., Ben-Zvi, R. and Schram, M. (1964) Arch. Biochem. Biophys. 104, 58--66 7 Batzri, S. and SeHnger, Z. (1973) J. Biol. Chem. 248, 3 5 6 - - 3 6 0 8 Dormer, R.L. and Ashcroft, S.J.H. (1974) Biochem. J. 144, 543--550 9 Leslie, B.A., Putney, J.W. and Sherman, J.M. (1976) J. Physiol. L o n d o n 260, 351--370 10 Riggs, D.S. (1970) The Mathematical Approach to Physiological Problems, Chapt. 6, E x p o n e n t i a l Growth and Disappearance, M.I.T. Press, Cambridge, MA 11 Frankenhaeuser, B. and Hodgkin, A.L. (1957) J. Physiol. L o n d o n 1 3 7 , 2 1 8 - - 2 2 4 12 Shanes, A.M. (1958) Pharmacol. Rev. 10, 59--164 13 Batzi, S., Selinger, A., Schramm, M. and Rabinovitch, M.R. (1973) J. Biol. Chem. 248, 361--368 14 Koelz, H.R., Kondo, S., Blum, A.L. and Schulz, I. (1977) Pfliigers Arch. 370, 37--44 15 Borowitz, J.L. (1972) Life Sci. 1 1 , 9 5 9 - - 9 6 4 16 Heuser, J. and Miledi, R. (1971) Proc. R. Soc. L o n d o n Ser. B, 179, 247--270 17 De Bassio, W.A., Schnitzler, R.M. and Parson, R.L. (1971) J. Neurobiol: 2, 263--278 18 Nayler, W.G. and Harris, J.P. (1976) J. Mol. Cell. Cardiol. 8, 811--822 19 Uyesaka, N., KalT1ino, K., Ogawa, M., Inouye, A. and Machida, K. (1976) J. Membr. Biol. 2 7 , 2 8 3 - 295 20 Kanagasuntheram, P. and Randle, P.J. (1976) Biochem. J. 160, 547--564