Amylase release from streptolysin fetal pancreatic acini

0 permeabilized

DANIEL J. CHER, PHILIP J. PADFIELD, AND JAMES of Cell Biology, Yale University School of Medicine, Department Cher, Daniel J., Philip J. Padfield, and James D. Jamieson. Amylase release from streptolysin 0 permeabilized fetal pancreatic acini. Am. J. Physiol. 262 (Gastrointest. Liver Physiol. 25): G719-G726, 1992.-Developmental regulation of Ca2+-dependent protein discharge was investigated in fetal and neonatal rat pancreatic acini permeabilized with streptolysin 0. When incubated at 37°C in a Ca2+-ethylene glycol-bis(Paminoethyl ether)-N,N,N’,N’-tetraacetic acid/K glutamate buffer, permeabilized day 19 and 20 fetal acini demonstrated Ca2+-dependent release of amylase, whereas day 21 (term) fetal acini did not. Ca”+-dependent amylase release reappeared in day 1,2, and 6 neonatal pancreases. ATP depletion completely inhibited Ca2+-stimulated amylase release from both day 20 fetal and adult acini. Ca2+-dependent amylase discharge from day 20 fetal acini was enhanced by the nonhydrolyzable GTP analogue, guanosine 5’-O-(3-thiotriphosphate) (GTP$S), and by the phorbol ester, 12-0-tetradecanoylphorbol 13-acetate (TPA). Ca2+ -independent GTPyS-stimulated amylase release was observed from adult but not from day 20 fetal acini. In contrast to its stimulatory effects in permeabilized adult acini, adenosine 3’,5’-cyclic monophosphate (CAMP) alone had little effect on release from permeabilized day 20 fetal acini. Our studies indicate that the fetal pancreas is competent to undergo Ca2+-dependent protein secretion but that this secretion is suppressed at birth. Our studies also suggest that the fetal gland is sensitive to modulators of exocytosis active in the adult pancreas, such as GTPrS, TPA, and CAMP but responds differently to these agents compared with responses in adult glands. secretion; pancreas; rat; development; 5’-O-(3-thiotriphosphate); adenosine phate; 12-0-tetradecanoylphorbol messengers

calcium ions; guanosine 3’,5’-cyclic monophos13-acetate; intracellular

MORPHOGENESIS AND CYTODIFFERENTIATION ofthe fetal rat exocrine pancreas are well-characterized processes. Secretory enzymes are detectable at day 12 of gestation (gestation is 22-23 days) (30) while structurally differentiated cells are first identifiable at day 17 (29, 33). By day 21, acinar cells show full structural (24, 29) and enzymatic (30) differentiation. The development of stimulus-secretion coupling in the fetal pancreas is less well-characterized. Previous work in our laboratory (6) and by others (8, 21, 38) has shown that pancreatic lobules prepared from fully cytodifferentiated term fetal rats do not discharge secretory proteins in response to either cholecytstokin (CCK) or carbachol despite the presence of their receptors on the plasma membrane (6, 8, 22). Our previous studies (7) also suggested that term rat pancreas is responsive to CCK and carbachol, but this has never been independently verified. Secretagogue responsiveness develops within the first 24 to 48 h of neonatal life (6, 21, 34, 37). The location of this signal-transduction “defect” in term fetal pancreas is controversial. Stimulation of adult pancreatic acini by CCK results in a rise in intracellular 0193-1857/92

$2.00 Copyright

D. JAMIESON New Haven, Connecticut

06510

Ca2+ and activation of protein kinase C (PKC; 13). An increase in intracellular [Ca”‘] elicited by ionophores has been reported to be capable (7, 38) or incapable (6) of stimulating amylase secretion from term pancreatic lobules. In 45Ca2+-loaded term fetal pancreatic lobules, 45Ca2+ efflux is stimulated by CCK (6), suggesting that Ca2+ mobilization is competent in the fetus. Even less is known regarding the role of protein kinases in signal transduction in fetal pancreas. Shimizu et al. (34) report that PKC is decreased in term-fetal and neonatal pancreas, rising to’ adult levels by day 2 of neonatal life. Day 2 pancreatic acini were responsive to 12-0-tetradecanoylphorbol 13-acetate (TPA; a PKC stimulator), whereas newborn acini were not. Dibutyryl adenosine 3’,5’-cyclic monophosphate (DBcAMP) stimulated amylase release from term fetal lobules alone (7, 38) or only in combination with A23187 (6). Of note, DBcAMP alone stimulated amylase release at day 7 of neonatal life in one report (7) but not at day 8 in another (38) Streptolysin 0 [SLO (a)] has been used extensively to study regulated secretion in semi-intact cells (1). The parameters for SLO permeabilization in adult pancreatic acini have been determined in our laboratory (28) and by others (9, 18). SLO permeabilization is advantageous because it is selective for the plasma membrane and allows passage of large or charged molecules into the cytoplasmic space. In the present study, we report a method for the preparation of viable pancreatic acini from fetal and neonatal rats at different developmental ages. We attempt to clarify the development of signal transduction in the pancreas by characterizing permeabilization of fetal pancreatic acini with SLO and studying the responses of permeabilized fetal and neonatal acini of different developmental ages to Ca2+ and other intracellular modulators of secretion. We find that Ca2+-dependent amylase secretion from SLO-permeabilized fetal acini is detectable in day 19 glands and decreases up to the time of birth. Ca2+-dependent secretion reappears within

the first

two days of neonatal

life.

Ca2+-dependent

secretion from permeabilized day 20 fetal acini is modulated by guanosine 5’-O-(3-thiotriphosphate) (GTPyS) and TPA but in a manner distinct from that in adult pancreatic acini. MATERIALS

AND

METHODS

Materials. Piperazine-N,N’-bis( 2-ethanesulfonic acid) (PIPES), Pronase E (from Streptomyces griseus), type IV DNase, fraction V bovine serum albumin (BSA), %deoxyglucase, carbachol, TPA, 4a-phorbol, and monopotassium glutamate were from Sigma Chemical (St. Louis, MO). N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES), collagenase P, ethylene glycol-bis(P-aminoethyl ether)-N,N,N’,N’-

0 1992 the American

Physiological

Society

G719

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tetraacetic acid (EGTA), antimycin A, ATP, CAMP, and GTPyS were from Boehringer-Mannheim (Indianapolis, IN). Soybean trypsin inhibitor (SBTI) was purchased from Calbiochem (La Jolla, CA) and 3-isobutyl-l-methyl xanthine was from Aldrich Chemical (Milwaukee, WI). Purified collagenase from clostridium histolyticum was from Worthington Biochemical (Freehold, NJ) and was used as described (28). Streptolysin 0, obtained from Burroughs- Wellcome Diagnostics (Triangle Park, NC), was reconstituted with distilled water at 20 IU/ml and stored at 4°C. Male and timed-pregnant female SpragueDawley rats were from Camm Research Laboratory Animals (Wayne, NJ). Isolation of pancreatic acini. Fetal rat pancreatic acini were prepared by a modification of Ref. 4. Timed-pregnant rats were killed by cervical dislocation, fetuses removed, and pancreata immediately dissected and placed in ice-cold buffer [(in mM) 140 NaCl, 4.8 KCl, 20 HEPES, 20 glucose, pH 7.41. Pooled whole glands (usually 6-8/litter) were transferred to a lo-ml siliconized Erlenmeyer flask containing 2 ml of modified KrebsRinger HEPES buffer (KRH) [(in mM) 140 NaCl, 4.8 KCl, 20 HEPES, 20 glucose, 2.0 CaC&,, 1.2 MgCIZ, 1 NaHPO* as well as 0.05% BSA and 0.01% SBTI, pH 7.4, preoxygenated for 30 min] with 0.5 mg/ml Pronase E and shaken at 120 oscillations/ min in a 37°C water bath for 10 min. Pancreata were transferred to a l&ml siliconized Corex tube, rinsed with KRH, and resuspended in 5 ml of KRH containing 0.25 mg/ml collagenase P. The tube was covered and shaken vigorously for 4 min, during which time the tissue dispersed. It was found that predigestion of the fetal pancreata with proteases was necessary for the preparation of viable acini. Without predigestion, a longer period of mechanical agitation and collagenase digestion was required for the tissue to disperse, resulting in cell clumps that were larger and thus inappropriate for permeabilization and less viable. At the beginning of the third minute of shaking, DNase was added to a final concentration of 1,000 U/ml. The mixture of acini and single cells was passed through a 2OO-pm Nytex filter and centrifuged for 2 min at 50 mean gravitational force averaged (9,“) to pellet acini. Acini were washed three times in buffer A (139 mM monopotassium glutamate, 20 mM PIPES, 0.01% SBTI, pH 6.6), and the final pellet was resuspended in -2 ml of permeabilization buffer B (see below). Buffer A was nominally Cl- free, because Cl- is low in acinar cell cytoplasm and, additionally, has been found by us to inhibit Ca2+-dependent secretion from permeabilized adult pancreatic acinar cells. The pH of bufferA was 6.6 to optimize Ca buffering by EGTA and to prevent zymogen granule lysis inside cells that will occur at pH values >7.0. Acinar viability, estimated by the exclusion of trypan blue, varied between 95 and 98% for day 21 acini and 97 and 99% for day 19 and 20 acini. Acini from adult glands were prepared similarly. The pancreas was removed from one 80-100 g adult male rat starved for 15-18 h, and acini were isolated as described for fetal glands with the following modifications. The pancreas was diced into l-mm cubes and transferred to a siliconized 15-ml Corex tube. Five milliters of isolation buffer containing 0.25 mg/ml collagenase P was added, and the tube was vigorously shaken for 10 min. Acini were filtered and washed as for fetal tissue except that the final acinar pellet was resuspended in 5 ml of buffer A. Viability by trypan blue exclusion was 97-99%. Acini from neonatal rats were prepared identically to the procedure described for adult male rats except that four to five neonatal pancreata were used, and the animals were suckled until just before being killed. Pronase E treatment was used for day 1 and 2 neonatal acinar preparations but not for day 6. Viability by trypan blue exclusion was -95% for day 1 neonatal acini and 97-99% for day 2 and 6 acini. Acinar permeabilization and assays for amylase release. For each secretion experiment, 50 ~1 of the acinar preparation was

FETAL

PANCREAS

placed in 2-ml tubes and 150 ~1 of buffer B containing 139 mM monopotassium glutamate, 20 mM PIPES, 0.01% SBTI, pH 6.6, EGTA (2 mM final), Mg2’ ATP (2 mM final), Ca”+, Mg2+, +SLO, and plus or minus indicated concentrations of intracellular modulators. Glucose was not present in any of the media after isolation of acini. [Ca”+] and [Mg2+] were calculated using a computer program provided by World Precision Instruments (New Haven, CT) (28) such that the final [Mg2+] was 2 mM, and the final [Ca”‘] varied between 10 nM and 100 PM. The tubes were capped and gently shaken at 37°C for times up to 25 min. The tubes were transferred into an ice-cold water bath for 2 min and then centrifuged for 2 min at 2,000 g,, in an Eppendorf microcentrifuge. One hundred microliter of supernatant was removed and saved. Amylase assay buffer (1.5 ml) containing 0.02% NP40 was added to the acinar pellet, and the cells were lysed by sonication (50 W) for 8 s. Supernatant and lysate were assayed for amylase according to the method of Bernfeld (3) using a modified assay buffer [(in mM) 50 TrisHCl, 2.0 CaC12, 20 NaCl, pH 6.91. The assay buffer contained CaC12, because the EGTA present in the samples, when assayed in Bernfeld’s phosphate-buffered saline, decreased the enzyme activity, presumably by binding Ca2’. Lactate dehydrogenase (LDH) was assayed according to Ref. 31. Amylase and LDH release are expressed as percent of total cellular enzyme. In a given experiment, 3 to 12 parallel incubations were carried out for each experimental condition; samples of supernatant and lysate were assayed for amylase or LDH once for each incubation. For time-course permeabilization experiments, acini were aliquoted into 2-ml plastic tubes and placed in an ice-water bath for 2 min. Ice-cold permeabilization buffers containing 0.5 IU/ml SLO were added, and the tubes were transferred to a 37°C water bath. At times between 0 and 25 min, the tubes were transferred back onto the ice-cold water bath. At the end of the incubation period, the acini were processed for percent amylase and LDH release. Metabolic inhibition. In experiments where acini were pretreated with metabolic inhibitors, acinar preparations were divided in half, washed three times with KRH, and resuspended in 10 ml of KRH containing 5 PM antimycin A and 20 mM 2deoxyglucose (omitting glucose) or in KRH with glucose and 0.1% dimethyl sulfoxide (vehicle in which antimycin A was dissolved). After incubation at 37°C for 30 min, noninhibited acini were collected, washed three times in buffer A (with no added glucose or ATP), and permeabilized with SLO in buffer B containing 2 mM ATP and again no glucose. Permeabilization buffer for metabolically inhibited acini was the same except that it lacked ATP; glucose was absent. Statistics. Statistical significance was tested using two-tailed Student’s t test for small independent samples. RESULTS

Response of fetal and adult pancreatic acini to carbachol. work in our laboratory and by others has shown that pancreatic lobules prepared from fetal rats 1 day before birth are unresponsive to CCK or carbachol, despite the presence of plasma membrane receptors for these hormones. We verified the lack of response to secretagogue in our fetal acinar preparation by incubating acini from day 20 fetal pancreata in the presence of various concentrations of carbachol and measuring the release of amylase (Fig. 1). Fetal acini were prepared using a variant of the procedure described in MATERIALS AND METHODS by omitting protease treatment and substituting purified collagenase for protease-contaminated collagenase P. This procedure was used to avoid proteoPrevious

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Fig. 1. Effect of carbachol on intact adult and fetal pancreatic acini. Acini prepared from day 20 fetal pancreata using purified collagenase or from adult glands using protease and collagenase treatments were incubated at 37°C for 25 min in Krebs-Ringer-HEPES buffer in presence of various concentrations of carbachol. Supernatants and lysates were assayed for amylase and release expressed as a percent of total cellular enzyme. Single experiments for day 20 fetal and adult acini are shown. Each data point represents mean t SD of amylase secretion assays carried out on 3 separate acinar cell incubations run in parallel.

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lytic digestion of cell-surface receptors. Acini produced by this method maintain good viability (>95% by trypan blue exclusion) but are too large for studies requiring extensive cell permeabilization. To control for the effects of protease treatment on the response to secretagogue, acini prepared from adult pancreas using Pronase treatment and protease-containing collagenase P were incubated with carbachol in parallel experiments where no difference in response to secretagogue was noted (not shown). Figure 1 shows that day 20 fetal acini are unresponsive to carbachol at concentrations that stimulate protease-treated adult pancreatic acini two- to threefold. Parallel experiments with day 21 fetal acini demonstrated a similar lack of response (not shown). Streptolysin 0-permeabilized fetal pancreatic acini. To investigate SLO permeabilization in the fetal pancreas, acini from day 20 (Fig. 2A) and day 21 (Fig. 2B) glands were incubated at 37°C for 25 min in complete permeabilization buffer (10 nM free Ca’+) containing various concentrations of SLO, and the release of amylase and LDH was determined. Large releases of LDH, a 140-kD cytosolic protein, in the context of small releases of amylase suggest that the plasma membrane but the not amylase-containing compartments is rendered permeant. In the absence of SLO, the release of LDH was ~5% for day 20 cells and -11% for day 21 cells. At an SLO concentration of 0.5 IU/ml, fetal acini released >55-58% of their intracellular LDH, and further increases in the concentration of SLO did not result in increased LDH release. Release of amylase was not increased in day 20 fetal acini in the presence of SLO, whereas SLO routinely increased the release of amylase in acini prepared from day 21 fetuses. The SLO titration curves shown are similar to those obtained for adult pancreatic acini prepared by the same method (28), with the exception that the release of LDH from adult cells reaches a plateau at -65-70% (not shown). In subsequent permeabilization studies, SLO was used at a concentration of 0.5 IU/ml. When incubated with 0.5 IU/ml SLO for 10 min in permeabilization buffer in the presence of 100 fig/ml

Fig. 2. Effect of streptolysin 0 (SLO) on lactate dehydrogenase (LDH) and amylase release in fetal pancreatic acini. A: acini from day 20 fetal pancreata were incubated in permeabilization buffer (free Ca2+ = 10 nM) at 37°C for 25 min with various concentrations of SLO. Results shown are means t SE of 2 identical experiments performed on consecutive days. In an individual experiment, single amylase secretion assays were carried out on 3 separate acinar cell incubations run in parallel. B: protocol as in A using acini from day 21 fetal pancreases. Means t SE of 2 consecutive experiments are shown.

ethidium bromide and visualized by fluorescence microscopy, day 20 fetal acini show staining of nearly 100% of their nuclei. In the absence of SLO, ethidium bromide stained ~5% of the cells (not shown). Ca2+-dependent amylase release from permeabilixed fetaZ pancreatic acini. Cell permeabilization using SLO (9,

18, 28) and other agents (17, 20) has allowed precise determination of the Ca2+ dependence of protein secretion from pancreatic acini. [Ca”‘] that stimulate maximum secretion from permeabilized adult acini (-10 PM) are 5- to lo-fold higher than reported for hormonestimulated increases in intracellular [ Ca2+] measured using fluorescent dye techniques (26, 35). Acini prepared from adult and fetal rat pancreata of different gestational ages were incubated in the presence of 0.5 IU/ml SLO in permeabilization buffer with free [Ca”‘] between 10 nM and 100 PM, and the release of amylase was determined (Fig. 3A). SLO-permeabilized adult acini released amylase in a Ca2+-dependent manner with a half-maximal [Ca”‘] of -5 PM and a maximal [Ca”‘] of -30 PM. This Ca2+ dose-response curve matches previous data from our laboratory (28) and is similar to that observed by others (9, 20). An identical experiment using acini from day 19 fetal pancreata showed a similar Ca2+ dose response except that the magnitude of the Ca2+-dependent response was approximately one-third that of the adult pancreas. A similar dose-response curve with a similar mean effective concentration (EC& was seen for permeabilized acini from day 20 fetal pancreata except that the Ca2+-dependent response was approximately one-half the day 19 response

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G722

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[Cal (MI Fig. 3. Developmental changes in Ca2+-dependent amylase release. Secretory responses to free Ca2+from adult pancreatic acini and from day 19, 20, and 21 fetal acini (A), and from day 1, 2, and 6 neonatal acini (B). Permeabilization of acini and secretion assays for amylase are described in METHODS. Data for day 19 acini are means ~fr SE from 2 separate experiments carried out on consecutive days. Data for day 20 and 21 glands are means k SE from 15 and 19 separate litters, respectively, examined on consecutive days, and a representative experiment out of 4 separate experiments is shown for adult glands. Data for days 1, 2, and 6 are from single litters *SD. For individual experiments at all ages, single amylase secretion assays were carried out on 3 separate acinar cell incubations run in parallel. Data for days 19 and 20 glands showed statistically significant (P < 0.001) differences between low (10 nM) and maximal (30 PM) free Ca2+ within a given age based on unpaired ‘Z-tailed Student’s t test. Within a given neonatal age, significant differences (P < 0.01) exist between low and maximal Ca2+ responses for days I, 2, and 6 acini. For clarity of presentation, data from various developmental ages are grouped in separate panels (A) and (B). F or comparison, data for adult acini are shown only in A. Ca2+ concentration curves are from separate experiments carried out on different days and so absolute values of secretory responses are not directly comparable.

PERMEABILIZED

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these cells. Although day 19 fetal acini also responded to Ca 2+, the number of acini generated from the small amounts of pancreatic tissue in day 19 fetuses was inadequate for routine study. The time course of Ca2+-dependent amylase release from day 20 fetal pancreatic acini was investigated during the first 16 min after permeabilization (Fig. 4). Permeabilization in the presence of 10 nM (nonstimulatory) free Ca2+ caused a small rise in amylase release with time, whereas permeabilization in the presence of 30 ,uM Ca2+ caused an even greater release with time (not shown). When the amylase-release data are corrected for secretion with IO nM Ca2+, the release curve shows that Ca2+dependent secretory response from day 20 acini was completed within the first minute after permeabilization. In contrast, experiments with acini from adult pancreas showed that Ca2+-dependent release of amylase is completed within -5 min after permeabilization. In subsequent experiments on fetal acini, the 37°C incubation period for permeabilization and secretory assays was shortened to 4 min. Ca2+-dependent release of amylase from permeabilixed fetal acini is permeabilization and ATP dependent. Metabolic inhibition blocks hormone-induced amylase secretion in the intact pancreatic acinar cell (15). In permeabilized adult rat acini, Ca2+-dependent amylase secretion occurs in the absence of exogenous ATP and is partially inhibited by brief (5-15 min) metabolic inhibition (P. J. Padfield, unpublished observations). The energy requirements for Ca2+-dependent amylase release from permeabilized fetal acini were studied by preincubating the cells in KRH with 2-deoxyglucose and antimycin A for 30 min, followed by permeabilization with SLO in the usual manner except that permeabilization and subsequent incubation media did not contain ATP and were glucose free (Fig. 5A). Fetal acini subjected to mock inhibition (KRH with glucose and dimethyl sulfoxide vehicle) and subsequently incubated in permeabilization buffer without SLO showed no Ca2’dependent increase in amylase secretion, presumably

7

and one-sixth of the adult response. Also, the doseresponse curve for day 20 acini exhibited a downturn at 100 PM Ca2+, similar to that for adult acini; this was not seen in day 19 acini. In contrast, SLO-permeabilized acini from day 21 fetal pancreata showed no Ca2’-dependent increase in amylase release. Possibly the lack of apparent response to Ca2+ may be due to the higher basal Ca2+-independent secretion from day 21 acini. Identical experiments examining Ca2+-dependent amylase secretion were performed using acini from day 1,2, and 6 neonatal pancreata (Fig. 3B). Permeabilized acini from day 1 neonatal pancreata showed small nonsignificant increases in secretion at [Ca”‘] >l PM. Permeabilized acini from day 2 and 6 neonatal rats showed small but significant Ca2’ -dependent secretory responses. Kinetics of amylase release from permeabilized fetal pancreatic acini. Because permeabilized acini prepared from day 20 fetal pancreata secreted amylase in response to lo-30 PM free Ca2+, we concentrated our studies on

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time (minutes) Fig. 4. Kinetics of Ca2’ -dependent amylase release from permeabilized fetal and adult acini. Acini from day 20 fetal or adult rat pancreata were permeabilized according to METHODS. Data plotted are result of subtracting release of amylase at 10 nM Ca2’ from release at 30 PM Ca2’ for each time point. Means t SE of 3 separate experiments are shown for fetal acini; means f: SE of secretory data from 3 separate adult acinar preparations are shown. In each experiment for day 20 and adult acini, single amylase secretion assays were carried out on 3 separate acinar cell incubations run in parallel.

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Fig. 5. Ca2+-dependent amylase release from permeabilized fetal and adult acini is energy dependent. Acini from day 20 fetal pancreata were metabolically inhibited (met inhib) or mock inhibited (mock inhib) as described in METHODS. A: means rt SE of results from 2 day 20 litters studied on consecutive days are shown. In an individual experiment, single amylase secretion assays were carried out on 3 separate acinar cell incubations run in parallel. *Significant difference (P c 0.01). B: data for adult glands are representative of at least 4 separate experiments. Each data point is mean t SD of single amylase secretion assays carried out on 6 separate acinar cell incubations run in parallel.

because the cells remain unpermeabilized and Ca2’ was unable to enter them. Acini subjected to mock inhibition and permeabilized in medium containing SLO plus 2 mM ATP (but lacking glucose as well) retained their responsiveness to 30 PM free Ca2+, although the magnitude of the response was less than seen in experiments without the 30-min pretreatment. In contrast, metabolic inhibition completely blocked the Ca2+-dependent release of amylase from SLO-permeabilized day 20 fetal acini despite an apparent increase in Ca2+-independent basal release. The reason for Ca2+-independent basal release with metabolic inhibition is unknown. Metabolic inhibition of acini prepared from adult glands using the same protocol followed by permeabilization with SLO also completely inhibited the Ca2+-dependent secretory response (Fig. 5B). In contrast to fetal acini, however, unstimulated secretion from metabolically inhibited adult acini was decreased. Thus ATP-depleting conditions that completely inhibit the Ca2+-dependent secretory response in adult acini also completely inhibit Ca”+dependent secretion of amylase from day 20 fetal acini, suggesting that Ca2+ -dependent amylase release from fetal acini is energy dependent. Fetal Pancreatic Response of Permeabilixed to Other Cellular Modulators

Acini

FETAL

PANCREAS

G723

(Fig. 1) and CCK but competent to mobilize Ca2+ in response to secretagogue (6), it was of interest to investigate the responses of permeabilized fetal acini to the signal-transduction coupling activator GTP+. Acini from day 20 fetal pancreata were permeabilized with SLO, exposed to low (10 nM), submaximal (10 ,uM), and maximal (30 PM) [Ca”‘] in the presence or absence of 100 PM GTPyS (28), and amylase release was determined (Fig. 6). At nonstimulatory [Ca”‘] (10 nM), GTPyS had no effect on amylase release from day 20 acini. At submaximal (10 PM) and maximal [Ca”‘] (30 PM), however, GTPyS caused an 105 and 70% increase, respectively, in Ca2+ -dependent secretion of amylase from permeabilized fetal acini. Parallel experiments using adult acini showed that GTPyS caused a 30 and 16% increase in amylase release at [Ca”‘] of 10 and 30 PM, respectively. In contrast to its effect on day 20 acini, with 10 nM Ca2+, GTPyS enhanced secretion by -60% in adult acini [see also (28)]. The data in Fig. 5 suggest that GTPyS-responsive elements are present in fetal as well as in adult pancreas. Response to TPA. Treatment of pancreatic acini with PKC activators such as TPA has been shown to cause amylase release in intact adult acini (11, 14, 25). Exposure of SLO-permeabilized adult acini to TPA in the presence of micromolar free [Ca”‘] has been shown by us (A. J. O’Sullivan and J. D. Jamieson, unpublished observations) and others (19) to stimulate amylase secretion, suggesting that PKC-dependent protein phosphorylation plays a role in modulating regulated secretion. We investigated the role of PKC in amylase secretion from fetal pancreas by permeabilizing day 20 acini at various free [Ca”‘] in the presence and absence of 0.1 PM TPA (Fig. 7). At 10 nM free [Ca”‘], TPA had no stimulatory effect on amylase secretion from permeabilized acini. At 3 and 10 ,uM free [Ca”‘], however, TPA increased release of amylase. At 30 PM free [Ca”‘], a dose that elicits maximal Ca2+-dependent amylase -release from fetal acini, on the other hand, TPA had no effect. These Ca2+-sensitizing effects of TPA in SLOpermeabilized fetal acini are similar to those seen in permeabilized adult rat pancreatic acini (A. J. O’Sullivan and J. D. Jamieson, unpublished observations) and those reported by Kitagawa et al. (19) in mouse pancreas, save for the stimulatory effect at 30 PM [Ca”‘], which was

3x1o-5 z z? 2

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Response to GTP+. GTPyS stimulates secretion from many cells including the pancreas (10). Our recent work (28) and that of others (9, 1819) suggests that the effect of GTPyS in stimulating Ca2+-dependent amylase release may result partially from its interaction with “classical” receptor-linked GTP-binding proteins and partially from a GTP-binding protein distinct from the “classical” receptor-linked G proteins termed GE. Because fetal pancreatic acini are unresponsive to carbachol

I

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Fig. 6. Response of pancreatic acini to guanosine 5’-0-(3-thiotriphosphate) (GTPqS). Acini from day 20 fetal pancreata were permeabilized with SLO in presence or absence of 100 ,uM GTP+, and release of amylase was determined. Data shown are means tSE of 3 separate experiments carried out on consecutive days. In an individual experiment, single amylase secretion assays were carried out on 3 separate acinar cell incubations run in parallel.

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% arnylase release

Fig. 7. Response of fetal pancreatic acini to 12-O-tetradecanoylphorbol 13-acetate (TPA). Acini from day 20 fetal pancreata were permeabilized with SLO in presence or absence of 0.1 PM TPA, and release of amylase determined. Data shown are means + SE of 3 separate experiments carried out on consecutive days. In an individual experiment, single amylase secretion assays were carried out on 6 separate acinar cell incubations run in parallel.

seen in adult but not in day 20 fetal acini. Fetal acini showed no response to the control substance 4cu-phorbol (0.1 PM) at 10 nM, 3 PM, or 30 PM [Ca”‘] (data not shown). Response to CAMP. In SLO-permeabilized day 20 fetal pancreatic acini, 100 PM CAMP (the maximal stimulatory dose in mouse acini) (19) had no effect at 10 nM Ca2+ and consistent but slight stimulatory effects at 10 and 30 PM [Ca”‘] in three separate experiments (data not shown). Identical experiments in SLO-permeabilized adult acini showed stimulatory effects of 100 PM CAMP at 3, 10 and 30 PM free Ca2+ but not at 10 nM Ca2+ (not shown; A. J. O’Sullivan and J. D. Jamieson, unpublished observations). Concentrations of CAMP from 0.1 PM to 100 PM had no effect on amylase release from SLOpermeabilized day 20 fetal acini at 3 PM free Ca2+, both in the absence and presence of the phosphodiesterase inhibitor 3-isobutyl-l-methyl xanthine (100 PM; not shown). DISCUSSION

We used cell permeabilization with SLO to study intracellular modulators of protein secretion in the developing rat exocrine pancreas. Permeabilization of adult pancreatic acini with SLO allows manipulation of the intracellular environment with preserved secretory responses to calcium and other second messengers (9, 18, 19,28). There are no reports in the literature concerning permeabilization of fetal rat pancreatic acini with SLO. The development of secretory responses to CCK and carbachol in the fetal rat exocrine pancreas lags behind structural development of the gland. Although little is known regarding the development of second messenger systems in fetal and neonatal pancreas, published reports are controversial. By permeabilizing fetal acini with SLO, we were able to investigate directly the effects of various modulators of secretion previously studied in the adult pancreas. We developed a modification of the technique of Bruzzone et al. (4) allowing rapid (45 min) preparation of functional acini from fetal and neonatal rat pancreases. Viability in these preparations exceeded 95% for day 21 fetal and day 1 neonatal acini and exceeded 97% for day 19 and 20 fetal and day 2 and 6 neonatal acini. We note that acinar cell viability seen in our preparations was

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higher than that reported for other neonatal acinar preparations (8590%) (37). Our studies verified that pancreatic acini prepared from rats at day 20 and 21 of gestation are unresponsive to the secretagogue carbachol at micromolar doses, which stimulated amylase secretion from protease-treated adult pancreatic acini two- to threefold. These results are in agreement with most reports except for a previous study of ours (7) in which term fetal pancreatic lobules (as opposed to acini) did secrete amylase in response to CCK and carbachol; we have subsequently not been able to replicate that study (Ref. 6 and current report). Using SLO as a permeabilization agent and a Ca2+EGTA/potassium glutamate buffer, we showed that acini prepared from fetal rat pancreata at day 19 and 20 of gestation release amylase in a Ca2+-dependent fashion, whereas acini from day 21 pancreata (term fetal) show no Ca2+-dependent release. Identical experiments on pancreatic acini prepared from day 1, 2, and 6 neonatal pancreata suggested that Ca2+-dependent release of amylase from SLO-permeabilized acini reappears within the first day of neonatal life. Although the magnitude of the Ca2+-dependent responses varied, the E& of each Ca2+dependent response was similar, suggesting that the Ca2+-sensitive exocytotic machinery is present in adult exocrine pancreas as well as in fetal and neonatal glands. We were unable to detect Ca2+-dependent release of amylase from permeabilized day 21 fetal acini in 19 separate preparations. This lack of response to Ca2+ contrasts with some (7, 38) but not other (6) studies. Of note, in these studies intact term fetal lobules and Ca2+ ionophores were used while the present study used SLOpermeabilized fetal acini. Possibly in day 21 glands Ca2+ mobilization is competent, but the machinery responsible for discharge and responsive to increased levels of intracellular Ca2+ is either incompetent or suppressed. As noted earlier, 45Ca2+efflux from fetal pancreatic lobules is responsive to secretagogues (6); those studies, however, did not allow a quantitative estimation of [Ca2+]i. In adult glands, direct measurements of intracellular [Ca2+]i correlate with secretagogue stimulation (e.g., 16, 27,36). Similar studies have not been done to our knowledge in developing pancreas. Such a study would be important to carry out to see if regulation of intracellular [Ca”‘] by secretagogues correlates with development and to elucidate the developmental regulation of potential homeostatic mechanisms for intracellular [ Ca2+] such as expression of protein kinases and calcium binding proteins (see, e.g., Refs. 6, 34 and references therein). Interpretation of the magnitude of the Ca2+-dependent secretory responses in our studies on permeabilized fetal and neonatal acini is complicated by the changing granule number and amylase content of the developing pancreas. As birth approaches, the percentage of the pancreas consisting of acinar cells increases, and the number of granules and amount of amylase per cell rises over loo-fold (24). Similarly, during the first 2 days of neonatal life the granule number and amylase content per cell decrease by -25fold (21). Finally, neonatal animals are constantly suckling, which alters their physiological state compared with fetuses. Although not directlv addressed in our studies, basal

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(23) from permeabilized acini (i.e., secretion at to increase as birth approached simultaneously with a decrease in Ca2+-dependent secretion and decreased during the first 2 days of neonatal life when Ca2+-dependent secretion reappeared. This increase in basal release is somewhat artificial in the setting of permeabilized cells (from which many intracellular modulators may leak out), and may reflect the l2% decrease in viability noted in day 21 fetal and day 1 neonatal acini (as opposed to earlier fetal or later neonatal ages). In this regard, basal secretion from permeabilized day 20 glands was reasonably constant among different experiments done at different times (ranging from -3.5-4%), which allows more confidence in the interpretation of results of individual experiments such as those in Fig. 5A where metabolic inhibition appears to elevate basal release. It is not inconceivable that Ca2+dependent secretion in the pancreas may be downregulated with concomitant increases in basal secretion at the time of birth when the animal is about to ingest its first nutrient-rich meal. In intact adult pancreatic acini, energy is required for secretion (15). In permeabilized adult rat pancreatic acini, Ca2+-dependent secretion takes place in the absence of exogenous ATP or glucose and is only partially inhibited by metabolic inhibition over lo-15 min with antimycin A and Z-deoxyglucose where ATP levels are reduced by >99% (Philip J. Padfield, unpublished results). In the experiments reported here, prolonged (>30 min) metabolic inhibition with antimycin A and Z-deoxyglucose in the absence of glucose and ATP throughout the permeabilization and Ca2+ exposure period confirmed that Ca2+-dependent amylase release from day 20 fetal as well as from adult acini is energy dependent, possibly indicating generation of ATP by respiration. We did not study the ability of glucose alone to support secretion from mock inhibited or metabolically inhibited permeabilized fetal acini. However, our previous studies (15) indicate that glycolytic inhibitors have little or no effect on secretagogue responsiveness in intact adult pancreatic lobules in vitro. Reversal of the effect of metabolic inhibition in permeabilized fetal acini by readdition of ATP was not examined in our study, because added ATP only partially (-40%) restores secretion from metabolically inhibited permeabilized adult acini (Philip J. Padfield, unpublished results) and so detection of a very small recovery might be difficult. For the future, it will be of interest to determine the relative contributions of glycolysis and respiration in supporting secretion from developing versus adult glands, because this knowledge could be important in understanding possible defects in pancreatic secretion and digestion that compromise growth in premature infants and infants with low birth weights. Amylase release from day 20 fetal acini was stimulated by modulators of second-messengers thought to play a role in exocytosis.’ For all three modulators tested, however, the effects differed from those obtained in experiments performed on adult acini. GTPyS increased amylase release in permeabilized day 20 acini at submaximal and maximal [Ca”‘] (10 and 30 PM), but showed no stimulatorv effect at low (10

10 nM [Ca2+]) appeared

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nM). In contrast, experiments with adult pancreatic acini showed stimulation by GTPyS at all [Ca”‘]. GTPyS has been shown to have both additive and Ca2+-sensitizing effects (28). The site of GTPyS action in the pancreas is not known but is postulated to act on phospholipase C-linked GTP-binding proteins associated with the CCK and muscarinic receptors and on cytosolic or granulebound GTP-binding protein(s) called GE (10, 28). Kitagawa et al. (19) have investigated the role of GTPyS and other intracellular modulators in Ca2+-dependent and independent secretion. Because day 20 fetal acini show no Ca2+ -independent GTPyS-stimulated secretion, some of the GTPyS-responsive elements controlling exocytosis in the adult pancreas may be missing in the fetal glands. TPA, a PKC stimulator, also demonstrated stimulatory effects in permeabilized day 20 fetal acini distinct from those seen in adult acini. In adult acini, TPA increased amylase secretion at 3, 10 and 30 PM [Ca”‘] but not at 10 nM (nonstimulatory) [Ca”‘], in agreement with our studies (O’Sullivan and Jamieson, unpublished observations) and those of others (18). In day 20 fetal acini, TPA stimulated amylase release at 3 and 10 PM Ca2+ but not at 10 nM or 30 PM Ca2+. We note that while Shimizu et al. (34) showed that PKC is significantly decreased in term fetal and neonatal (day I ) pancreas and that TPA at 0.1 and 1 PM had no effect on amylase release from intact neonatal pancreatic acini, we were able to show an effect of 100 nM TPA in day 20 fetal acini. TPA-stimulated amylase release from term fetal and day 20 acini was not tested in the report of Shimizu et al (34). Our results suggest that PKC activation plays a role in Ca2+ -dependent exocytotic events in the fetal pancreas as in the adult pancreas, but that other interacting factors may differ. Activation of adult pancreatic acini by vasoactive intestinal peptide and secretin causes increases in intracellular levels of CAMP with subsequent activation of protein kinase A (32). Calcium and CAMP stimulate protein secretion synergistically in intact acini (5,12,32) In permeabilized acini the effects of CAMP are unclear (17, 19, 20). In fetal pancreas, the role of CAMP in secretion is less well-defined. CAMP analogues stimulate amylase release from intact term fetal lobules alone (7, 38) or only in combination with A23187 (6). In SLOpermeabilized day 20 acini, CAMP had small stimulatory effects on amylase release. Parallel experiments on permeabilized adult acini and similar experiments by other investigators [see Ref. 19, also at 100 PM CAMP] showed CAMP-stimulated amylase release. Perhaps in day 20 fetal acini, the CAMP-mediated path.way for amylase release is not fully developed or is lacking in crucial component(s) that account for the the small secretory response to CAMP alone. In summary we have used cell permeabilization with SLO in fetal rat pancreatic acini to demonstrate that Ca2+-dependent amylase release decreases as birth approaches and increases after birth. Ca2+-dependent amylase release from fetal glands is completed in a shorter time than in adult pancreas, and is modulated by second messengers in ways distinct from those in adult pancreas. Further study of difference in responses of fetal and

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adult pancreata may help define the roles of intracellular modulators. The authors acknowledge the critical reading of this paper by Dr. Antony O’Sullivan and the help of Henry Tan in preparation of material for publication. This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-17389 to J. D. Jamieson. D. J. Cher is a 1990-1991 Research Fellow of the National Kidney Foundation, New York, NY. Address for reprint requests: J. D. Jamieson, Dept. of Cell Biology, Yale Univ. School of Medicine, 333 Cedar St., New Haven, CT 06510. Received

28 May 1991; accepted in final form 29 October

1991.

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