WI; bovine serum albumin, Fraction. V, from Miles Laboratories, ..... Cell BioZ. 66, 165-181. 19. Czech, M. P., Lynn, D. G., and Lynn, W. S. (1973) J. BioZ. Chem.
THE JOURNAL OF B~LOCICAL Vol. 254, No. 16, Issue of August Prrnled m II SA.
Stimulation Pancreatic
CHEMISTRY 25, pp. 7624-7629.
1979
of the Glucose Transport Acini by Cholecystokinin
System in Isolated and Analogues*
(Received
Murray
Korc&j
From the Departments Metabolism Section,
John
A. Williams,
for publication,
July
Mouse
14, 1978, and in revised
form,
April
26, 1979)
and Ira D. GoldfineS
of Medicine and Physiology, University of California Service,
University Veterans
Cholecystokinin and analogues increased the uptake of 2-deoxy-n-glucose and 3-0-methylglucose into isolated mouse pancreatic acini. This uptake was mediated by a facilitated glucose transport system that was saturable, stereospecific, and was inhibited by both phloretin and cytochalasin B. In agreement with previous studies of acinar function, caerulein was more potent and pentagastrin less potent than cholecystokinin in increasing sugar transport. The choline&c analogue carbachol mimicked the effect of caerulein; atropine completely abolished the effects of carbachol but was without influence on the effects of the polypeptide hormones. In contrast, secretin, as well as dibutyryl cyclic AMP and dibutyryl cyclic GMP, had no effect on 2-deoxy-D-glucose uptake. Two lines of evidence suggested that hormonal stimulation of this sugar transport system was related to mobilization of cellular Ca’+. First, depletion of cellular Ca2+ by incubation of acini with ethylene glycol bis(&aminoethyl ether) N,N,iV’,iV’-tetraacetic acid (EGTA) reduced the effect of caerulein. Second, the Ca2+ ionophore A23187 mimicked the effects of caerulein on 2-deoxy-D-glucose uptake when Ca2+ was present in the medium.
Cholecystokinin (CCK) and analogues are potent stimulators of pancreatic amylase release (1,2). Evidence from several types of experiments indicates that the effects of these hormones on amylase release are mediated by the mobilization of Ca2’ from intracellular stores (3). First, the Ca2+ ionophore A23187, a compound known to increase intracellular CazC levels, mimics the secretory effects of these hormones (4, 5). Second, CCK’ markedly increases 45Ca2’ efflux (6-8). Finally, studies employing the fluorescent probe chlorotetracycline, an agent used to localize membrane-bound divalent cations, demonstrate that the CCK analogue caerulein induces the release of bound intracellular Ca2+ (9). It is not known, however, whether other cellular effects of CCK and analogues on pancreatic acini are also mediated through Ca’+. We now find that both CCK and caerulein * This investigation was supported by the Medical Research Service of the Veterans Administration and by Grants AM-19415, AM21089, and GM-19998 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC. Section 1734 solely to indicate this fact. 3 To whom reprint requests should be addressed at the Department of Physiology, University of California, San Francisco, California 94143. ’ The abbreviations used are: CCK, cholecystokinin; 2DG, 2-deoxyD-gh.ICOSe; 3-O-MG, 3-0-methylglucose; Hepes, 4-(2-hydroxyethyl-lpiperazineethanesulfonic acid; EGTA, ethylene glycol bis@-aminoethyl ether) N,N,N’,N’-tetraacetic acid.
of California, Administration
San Francisco, California Hospital, San Francisco,
94143 and California
the 94121
stimulate the uptake of the glucose analogues 2-deoxy-D-glucase (2DG) and 3-0-methylglucose (3-O-MG) into isolated mouse pancreatic acini. We investigated, therefore, the properties of this transport system and the role of Ca2+ in the hormonal regulation of this function. EXPERIMENTAL
PROCEDURES
Materials-Soybean trypsin inhibitor type l-S, luciferin-luciferase, hexokinase, hyaluronidase type 1, carbamylcholine (carbachol), antimycin, 30methylglucose, 2-deoxy-n-glucose, L-glucose, phlorizin, dibutyryl cyclic AMP, dibutyryl cyclic GMP, and 8 Br-cyclic GMP were purchased from Sigma Chemical Co., St. Louis, MO; chromatographically purified collagenase and chymotrypsin, from Worthington Biochemicals, Freehold, NJ; caerulein and pentagastrin, from Calbiochem, San Diego, CA, [“H]2-deoxy-n-glucose (specific activity, 8.26 Ci/mmol), [3H]3-O-methylglucose (specific activity, 80.8 Ci/mmol), and 45Ca2+ (specific activity, 22.7 mCi/mg), from New England Nuclear, Boston, MA; minimal Eagle’s medium amino acid supplement from GIBCO, Grand Island, NY; cytochalasin B and phloretin, from Aldrich Chemicals, Milwaukee, WI; bovine serum albumin, Fraction V, from Miles Laboratories, Elkhart, IN, and from Reheis, Chicago, IL. Purified porcine CCK and secretin were obtained from Gastrointestinal Hormone Research Unit, Karolinska Institutet, Stockholm, Sweden. Purified rat growth hormone was a gift from Dr. Jesse Roth, National Institutes of Health. Ionophore A23187, generously donated by Dr. Robert Hamill of Eli Lilly, was dissolved in 100% ethanol at a concentration of 2 mM. In all experiments using ionophore, comparable amounts of carrier ethanol were added to the control samples. In no case was more than 0.5% ethanol added and this amount had no effect on basal or caerulein-stimulated glucose transport or amylase release. Buffers-To prepare isolated acini and perform amylase secretion and Ca2+ efflux studies, Krebs-Henseleit bicarbonate buffer, and Hepes-Ringer buffer containing 5.6 mM glucose were prepared as previously described (IO, 11) and enriched with minimal Eagle’s medium amino acid supplement, 0.1% soybean trypsin inhibitor, 1% bovine serum albumin, and 2 mM sodium pyruvate. Experiments on 2DG and 3-O-MG uptake employed Hepes-Ringer without glucose and contained 0.5% albumin that had low insulin-like activity. Preparation ofdcini-Six to eight male white Swiss mice weighing 18 to 24 g were fasted 14 to 18 h prior to killing by decapitation. The pancreases were then rapidly removed and 1 g was injected with 5 ml of Krebs-Henseleit buffer with Ca”+ reduced to 0.1 mM and, in addition, 60 to 75 units/ml of collagenase, 20 to 40 pg/ml of chymotrypsin, and 1.8 mg/ml of hyaluronidase. Following a lo-min incubation at 37”C, shaking 120 rpm, the enzyme solution was replaced with 5 ml of fresh enzyme solution. The pancreases were then reincubated for 40 min and dissociated by forceful pipetting through polypropylene pipettes with decreasing orifices. Filtration through 150-p mesh nylon cloth was followed by centrifugation through Krebs-Henseleit buffer containing 4% bovine serum albumin. The acini were washed twice with the same buffer and then once in Hepes-Ringer buffer, after which they were preincubated in polycarbonate flasks for 60 min in Hepes-Ringer at 37”C, shaking at 60 rpm. All studies were performed at an acini concentration of 0.5 to 1.1 mg of protein/ml. The preparations obtained were isolated acini consisting of approximately 30 acinar cells that maintained their original polarity around an intact lumen (11, 12). For measurements of amylase secretion, the acini were incubated
7624
Glucose
Transport
by Pancreatic
in 25-ml polycarbonate Erlenmeyer flasks containing 2 ml of fresh Hepes-Ringer buffer. Hormones were then added, and the amylase released into the supernatant over the next 30 min was measured as previously described (lo), utilizing the assay procedure of Rinderknecht et al. (13). For the [3H]2DG uptake experiments, after the preincubation period, acini were incubated in 3 ml of fresh glucose-free HepesRinger buffer allowing lo-min recovery. Hormones were then added; 10 min later, r3H]2DG (1 pCi/ml) without carrier 2DG was added. The incubation was terminated at specified times by adding 0.7-ml aliquots to 10 ml of ice cold 0.9% saline, followed by immediate suction through Nuclepore filters (3-p pores) and a rinse with 5 ml of cold saline; the entire filtration process took approximately 10 s. Acini were removed from the filter and disrupted in distilled water by sonication. A sample was removed for protein assay employing a bovine serum albumin standard (14), and radioactivity of an aliquot was measured in a liquid scintillation counter (11). Total medium radioactivity was also measured in each incubation flask in order to compute 2DG uptake, expressed as pmol/mg of protein. Incubation
and processing of acini for r3H]3-O-MG
uptake experiments
were
carried
out as for [3H]2DG experiments, except for the concomitant addition of 0.1 or 1.0 mM carrier 3-O-MG at the time of addition of [3H]3-0-MG. With YSsucrose as an extracellular marker, it was
determined that trapped extracellular
fluid was negligible (less than
0.1 pl/mg of protein). For %a*+ efflux
experiments, preincubation was performed for 60 min with media containing 2 $i/ml of %a*+. At the end of this preincubation period, the acini were centrifuged in precooled tubes at 50 x g for 2 min, washed with ice cold Hepes-Ringer buffer, centri-
fuged, and resuspended in fresh Hepes-Ringer buffer at 37°C. Aliquots of ““Ca*+-loaded
acini
were
removed
immediately
(time
zero)
period,
the acini
were collected
by filtration
and treated
as for
[3H]2DG experiments. Paper chromatography of trichloroacetic acid-soluble extracts from sonicated acini was carried out on Whatman 3MM paper (15). The solvent mixture consisted of 30 ml of 1 M ammonium and 70 ml of ethanol (95%). Standards of [3H]2DG
acetate (pH and [3H]2DG-6-
5)
phosphate prepared using hexokinase were run in parallel with the unknown samples. Chromatographs were cut into strips and radioactivity was eluted with water and counted in a liquid scintillation counter. Cellular ATP levels were measured by the method of Stanley and Williams (16), employing luciferin-luciferase firefly extract. After
incubation as described for amylase release studies, acini were centrifuged;
the pellet
was washed
quickly with cold 0.9% NaCl tracted by sonicating in 1 ml of 0.5 N cold perchloric acid. Unless otherwise stated, the data shown are representative
and exof three
or more separate experiments. RESULTS
Hormonal Effects on c3HJ2DG
Uptake-CCK
3’0
bb Minutes
alogues caerulein and pentagastrin stimulated the uptake of r3H]2DG into isolated mouse pancreatic acini (Table I, Fig. 1A). Caerulein was the most potent compound, producing half-maximal effects at 0.1 to 0.2 rig/ml (approximately 0.1 nM) and maximal effects at 1 to 3 rig/ml (approximately 1 nM) (Fig. 1B). CCK was 10% as potent as caerulein (Table I), whereas pentagastrin was a much weaker agonist than CCK. The relative potencies of these three analogues in stimulating [3H]2DG uptake are in agreement with their established effects in stimulating enzyme secretion both in viva and in vitro (1, 11). In contrast, growth hormone, which in vitro has no effect on amylase secretion, secretin, which increases pancreatic fluid secretion, and the cyclic nucleotides dibutyryl cyclic AMP and dibutyryl cyclic GMP had no effect on [“H]BDG transport. Because caerulein was both more potent and more readily available than CCK, it was used in subsequent studies. In order to exclude the possibility that caerulein or other agonists stimulating glucose transport affected 2DG uptake through an effect on sugar phosphorylation, the percentage of label present in acini as 2DG or its metabolites was studied. Paper chromatography of trichloroacetic acid-soluble radioactivity revealed only two peaks of radioactivity corresponding to 2DG and 2DG-6-phosphate. As shown in Table II, neither submaximal nor maximal concentrations of caerulein, or the ionophore A23187, altered this percentage of 2DG
and
collected on Nuclepore filters. Additional aliquots were added rapidly to 25-ml Erlenmeyer flasks containing caerulein. After a 5-min incubation
7625
Acini
and its an-
do
TABLE
Effects
I
of hormones
on r3H]2DG uptake by isolated mouse pancreatic acini Specified additions were made 10 min prior to addition of [“H]BDG (1.0 x 10m7 M) and acini were collected 30 min later. All values are the
mean f SE. of triplicate determinations. Additions
[“H]2DG pmol/mgprotein/30
None
Per cent crease
uptake
in-
90
min
0.31 f 0.03
Caerulein (lo-” M) ( 1o-9 M) Cholecystokinin ( 1o-9 M) (I@
M)
Pentagastrin (3 X 1o-6 M) Growth hormone (1o-6 M)
0
.Ol
0.71 f 0.10
127
0.91
+ 0.13
190
0.73 f 0.12 0.85 f 0.10
132
0.67 f 0.04
114
0.30 * 0.01
0
.03 Caerulein
.l
172
.3
1
3
(rig/ml)
1. Effect of caerulein on [3H]2DG uptake by mouse pancreatic acini. A, time course; caerulein concentration, 1 rig/ml. B, doseresponse; incubation was terminated at 30 min. All values are the mean + S.E. of triplicate determinations. In this and subsequent experiments, the concentration of [3H]2DG was 1.0 x 10m7M. FIG.
Glucose
7626
Transport
phosphorylated by acini. Caerulein also stimulated the uptake of the nonmetabolizable glucose analog 3-O-MG (Fig. 2), providing additional evidence that the effect of this group of hormones on 2DG uptake is independent of intracellular phosphorylation. Paper chromatography (not shown) demonstrated that the intracellular [3H]3-0-MG was unaltered. Characteristics of Basal and Hormone-Stimulated r3H]2DG Uptake; Evidence for Enhancement of Facilitated Transport-Both control and stimulated [3H]2DG uptake were linear for up to 45 min (Fig. 1A). When 2DG uptake at 30 min was studied as a function of the 2DG concentration in the medium, both saturable and nonsaturable (linear) components were present (data not shown). The saturable component presumably represented carrier-mediated uptake; the
Effects Values min.
TABLE II on 2DG uptake and subsequent phosphorylation of duplicate determinations of uptake
by Pancreatic Effects
Acini of various
All values are the uptake at 30 min. Additions
TABLE III sugars on r3HJ2DG uptake by isolated pancreatic acini mean f S.E. of triplicate determinations T”HlPDG
untake
pmoUmgprotein/30
None L-Glucose 5mM 15I’XIM D-Glucose 5mM 15DIM 30Methylglucose 5mM 15mM
of
min
0.81 + 0.04 0.82 f 0.11 0.89 +- 0.06 0.21 + 0.04 0.13 + 0.01 0.49 * 0.03 0.38 + 0.05
of secretagogues
are the mean
[3H]2DG pmol/mg/30
None Caerulein 1 w/d 0.1 rig/ml A23187 (5
/AM)
2DG-6-P
uptake
at 30
(Per cent total)
min
w
0.39
84
1.40 0.88 0.97
83 86 84
I 10 -7
10
I -6
10 InhIbitor
I
0
I
I
I
2
i
4
I
1 -5
10
-4
,o
-L
(M)
;
MINUTES
FIG. 2. Effect of caerulein (1 rig/ml) on the uptake of [%]30-methylglucose by mouse pancreatic acini. The zero time uptake is equivalent to the trapped volume determined by using [‘4C]sucrose. Values are the means of duplicate determinations of uptake in the presence of 1 mM 30methylglucose.
Cytochalasm
B (M)
FIG. 4. Effect of inhibitors on basal and caerulein-stimulated [‘H]2DG uptake by mouse pancreatic acini. A, effect of phlorixin, phloretin, and cytochalasin B on basal glucose transport. B, effect of cytochalasin B on basal and caerulein-stimulated (1 rig/ml) 2DG uptake. Values are the means of duplicate determinations of uptake at 30 min. -0 2
02
04 l/2
0.6
06
IO
DG (mM)-’
FIG. 3. Double reciprocal plot of the saturable component of C3H]2DG uptake by unstimulated and caerulein-stimulated (1 rig/ml) mouse pancreatic acini. The contribution to transport by the linear component was measured and subtracted. Uptake was determined at 30 min and the values presented are the means of duplicate determinations. In this experiment, nonradioactive 2DG was added at specified concentrations.
linear component presumably represented diffusion. Double reciprocal plots of the saturable component (Fig. 3) revealed that caerulein increased the transport V,,,., without an effect on the transport K, (which was 10 111~). Other sugars in addition to 2DG interacted with this transport system. D- but not L-glucose profoundly inhibited [3H]2DG uptake; 3-O-MG had a significant but smaller effect (Table III). This sugar transport system was also influenced by known inhibitors of
Glucose
Transport
by Pancreatic
Acini
7627
)-
p
yr ‘, ,
01
I
I
I
03
10
30
I
I
IO
30
//
I 1o-6
0
Carbachol(fiM)
5. Effect atropine. Uptake FIG.
of carbachol was determined
I W5
lonophore
(M)
(A) and ionophore A23187 (B) on uptake of C3H]2DG by mouse pancreatic at 30 min and the values presented are the mean f SE. of triplicate determinations. TABLE
,A Y
1.6-
2 5
1.2-
g c-4 F -0
.8-
acini,
0, plus
IV
Effect of extracellular Ca’+ on r3HJ2DG uptake Following 60-min preincubation in medium containing 1.28 mM Ca*+, acini were centrifuged and resuspended in normal medium or in medium without Ca*+ and with 0.1 mM EGTA. Caerulein or A23187 additions were made after 10 min, followed 10 min later by addition of [3H]2DG. Uptake was determined at 30 min and the values presented are the mean -+ S.E. of triplicate determinations.
A-
[“H]LDG Additions ca*+
present
uptake EGTA
pmol/mgprotein/30
0.70 + 0.02 1.86 + 0.14 1.03 * 0.02
None Caerulein (1 rig/ml) A23187 (1 PM)
Effects of Carbachol cholinergic
FIG. 6. Effect of caerulein and ionophore A23187 on both [‘H]2DG uptake and amylase release. A, [3H]2DG uptake. Caerulein (1 rig/ml) and ionophore A23187 (5 pM) were studied alone and in combination. Uptake is pmol/mg of protein/30 min. B, Amylase release. Caerulein (0.1 rig/ml) and ionophore A23187 (1O-6 M) were studied alone and in combination. Amylase release is expressed in units/mg of protein/30 min. All values are the mean f SE. of triplicate determinations.
facilitated glucose transport (Fig. 4A). Both the potency values for these agents (phlorizin < phloretin < cytochalasin B) and the concentrations required for 50% inhibition of uptake were similar to values reported for these agents in other tissues (17-19). In addition, cytochalasin B (Fig. 4B) inhibited hormone-stimulated 2DG uptake. Neither control nor caerulein-stimulated 2DG uptake was inhibited when Na’ was replaced by choline in the incubation medium (data not shown). The above studies indicated, therefore, that hormones increase 2DG uptake through a facilitated transport mechanism.
3 PM
agents
and
and Ionophore the
ionophore
0.68 -+ 0.04 1.22 f 0.02 0.76 + 0.03
A23187-Since A23187
present min
stimulate
both amy-
lase release from isolated acini (II), their effects on [3H]2DG uptake were also studied. The cholinergic agonist, carbachol, produced half-maximal stimulation of [3H]2DG uptake at 2 pM and maximal stimulation at 30 PM (Fig. 5A). Atropine at 3 PM completely blocked carbachol stimulation but had no effect on either basal or caerulein-stimulated uptake. Ionophore A23187 also increased [3H]2DG uptake, maximal stimulation occurring at 5 PM (Fig. 5B). Adding the Ca2+ ionophore A23187 to maximally stimulating concentrations of caerulein did not further increase amylase release (Fig. 6B). Similar studies were performed with [3H]2DG uptake. When ionophore was added to caerulein, there was no further increase in [3H]2DG uptake (Fig. 6A). Relationship of r3H]2DG Uptake to Ca’+-Incubating acini in the absence of Ca” and in the presence of 0.1 mM EGTA did not influence control [3H]2DG uptake (Table IV). While the effect of caerulein was reduced by approximately 50%, the effect of ionophore was nearly abolished. These results are consistent with a number of studies on amylase release showing that the stimulating effects of A23187 are totally dependent on the presence of extracellular Ca2’, whereas hormone or cholinergic-stimulated release are initially independent of extracellular Ca2+ but become reduced upon depletion of intracellular Ca2+ stores (3, 20). Since a primary effect of pancreatic secretagogues is to mobilize intracellular Ca2+, we studied this mobilization by employing acini preloaded with 45Ca2+. Efflux of 4’Ca2+ from isolated acini, reflecting intracellular mobilization of Ca*+, falls
7628
Glucose
0
01
03
1
3
CAERULEIN
TABLE
1
3
(ng ‘ml)
FIG. 7. Relationship of ‘%a’+ efflux [3H]2DG uptake by mouse pancreatic 45Ca*1 &flux (5 min after adding caerulein) scribed under “Experimental Procedures.” 11%/5 min. Maximally stimulated efflux amylase release was 3.8%, and maximally 14.5% of total amylase content. Control pmol/mg of protein/30 min, and maximal of protein/30 min. All curves represent separate experiments in which values were of maximal stimulation.
Effects
Transport
to amylase release and acini. Measurements of were performed as deControl 45Ca2+ efflux was was 68%/5 min. Control stimulated amylase was [3H]2DG uptake was 0.53 uptake was 1.54 pmol/mg the mean f S.E. of four normalized as a percentage
V
of stimulators
and inhibitors ofpancreatic amylase release on acinar ATP content ATP content was measured 30 min after the specified addition. All values are the mean f S.E. of the number of observations in parentheses. Additions
ATP
None Antimycin (@
Content
nmol/mgprotein 28.5 f 0.4 (6) M)
Caerulein (0.1 w/ml) (1.0 w/ml) Carbachol (3 X lo-” M) A23187 (1 X lo-”
M)
into two kinetic components. loss from a rapidly exchanging effect at 5 min; these results
1.3 + 0.04
(3)
26.8 rt_ 1.2 34.6 f 1.2
(6) (6)
34.3 -+ 2.0
(6)
27.2 + 1.1
(6)
Caerulein greatly enhanced the compartment, with a maximal are similar to those previously
seen using isolated acinar cells (8). A significant increase in 45Cazt efflux occurred at a caerulein concentration of 0.01 ng/ ml and maximal effects were seen at 0.3 rig/ml (Fig. 7). The dose-response curve of caerulein-stimulated [3H]2DG uptake paralleled that of 45Ca2+ efflux but required 2- to a-fold higher concentrations of caerulein to produce equivalent effects. In contrast, maximal stimulation of amylase release was achieved with concentrations of caerulein lower than needed to maximally increase ?a2’ efflux; higher concentrations of caerulein inhibited amylase release (11). ATP Levels-To rule out the possibility that stimulated [3H]2DG uptake occurred as a result of lowered ATP stores (21,22), cellular ATP levels were measured (Table V). Neither caerulein, carbachol, nor ionophore A23187 lowered ATP levels. Furthermore, concentrations of caerulein and carbachol that were maximal for [3H]2DG transport slightly increased cellular ATP levels. In contrast, antimycin, a potent inhibitor of electron transport, lowered ATP content to 5% of control. DISCUSSION
The present data suggest that the pancreatic acinar cell possesses a transport system for glucose. Both 2DG and 3-0MG were employed as probes of this system. That sugar
by Pancreatic
Acini
uptake was mediated through a facilitated transport system was based on our findings that the system exhibited saturation kinetics, glucose stereospecificity, and inhibition by cytochalasin B, phloretin, and phlorizin. Our data also indicated that Ca2+ may function as an intracellular mediator of CCK-stimulated glucose transport in pancreatic acini. We find that: 1) the calcium ionophore A23187, a compound that facilitates the entry of calcium into acinar cells (5, 23), increases the uptake of 2DG into isolated pancreatic acini; 2) CCK and its analogues caerulein and pentagastrin, and the cholinergic analogue carbachol, substances that increased pancreatic enzyme release by increasing intracellular Ca2+ (3), all increased 2DG uptake; 3) the response to maximally stimulating concentrations of A23187 and caerulein was not additive; and 4) removal of Ca2+ from the medium and addition of EGTA (which depletes most of the extracellular and some of the cellular Ca’+) both inhibited the stimulation of 2DG uptake by ionophore A23187 and decreased the effect of caerulein. Most likely, caerulein was able to increase 2DG uptake despite extracellular Ca2+ depletion, because of the known ability of caerulein to release Ca” from intracellular stores. Studies in other tissues have indicated that intracellular Ca2+ may regulate glucose transport. Holloszy and Narahara (24) suggested that increased intracellular Ca2+ stimulates glucose transport in contracting muscle. The stimulation of glucose transport in muscle by anaerobic conditions and mitochondrial uncouplers has also been ascribed to an elevation in cytoplasmic Ca2+ levels (22). More recently, it has been shown that the ionophore A23187 stimulates glucose uptake into both thymocytes (25) and chick embryo heart cells (26). In addition to supporting the previous data indicating that Ca” regulates cellular glucose transport, our present findings in acini suggested that the effect of Ca2’ on glucose transport may be under hormonal regulation. Although hormone stimulation of both glucose transport and amylase release is presumed related to a rise in the level of cytoplasmic Ca2+, the dependence of these two cellular functions on Ca2+ may be different. Since, in acini and other tissues, cytoplasmic Ca2+ levels cannot be measured directly, we measured the enhancement of 45Ca2’ efflux by the CCK analogue caerulein. Because CCK and analogues do not increase Ca2+ influx (6,8,20), increased 4aCa2+ efflux reflects the release of stored Ca2+ into the cytoplasm and then transport out of the cell. The stimulation of 45Ca2’ efflux by caerulein was detectable at 0.01 rig/ml and maximal at 0.3 rig/ml; there was no additional increase in efflux at higher concentrations of hormone. Stimulation of amylase release was detectable at 0.01 rig/ml and maximal at 0.1 rig/ml. At higher concentrations of hormone, however, there was a decrease in amylase release. These findings suggest that amylase release is stimulated by small increases in cytoplasmic Ca2+ levels and is partially inhibited at greater levels. In contrast, caeruleinstimulated 2DG transport was slightly less sensitive to rises in cytoplasmic Ca2’, but increased to a maximum in parallel with YJa2+ efflux. The present studies allow construction of a model whereby CCK and related hormones may produce coordinated responses in pancreatic acinar cells. CCK and its analogues stimulate events at the acinar cell surface including glucose transport and membrane depolarization, and events in the cell interior including enzyme synthesis and release, glucose and amino acid metabolism, and nucleic acid and phospholipid synthesis (2). Since binding sites for the CCK analogue, caerulein, have been demonstrated on acinar cell plasma membranes (27) and the effects of CCK on intracellular functions such as amylase release occur within minutes, it is the consen-
Glucose
Transport
sus that CCK acts via a second messenger system. Although early reports suggested that cyclic AMP was the second messenger for CCK, it is now clear that cyclic AMP does not mediate the actions of this hormone (28, 29). CCK and analogues have been shown to increase cyclic GMP levels in pancreatic acini, but this rise in cyclic GMP is secondary to a rise in intracellular CaZf (23). Furthermore, our results using exogenous cyclic GMP analogues suggest that cyclic GMP does not mediate the rise in 2DG uptake observed with caerulein. Since the present study indicates that intracellular calcium may also mediate hormone-stimulated glucose transport as well as enzyme release, it is possible that changes in calcium levels may mediate both the cell-surface and intracellular functions of CCK. Acknowledgments-We thank the members of the Endocrine and Metabolism Sections of the San Francisco Veterans Administration Hospital for their critical review of this manuscript and Dr. R. L. Dormer for assistance with the chromatographic separation of radioactive sugars and metabolites. REFERENCES 1. Jorpes, J. E., and Mutt, V. (1973) in Secretin, Cholecystokinin, Pancreozymin and Gastrin (Jorpes, J. E., and Mutt, V., eds) pp. 1-179, Springer-Verlag, Berlin 2. Webster, P. D., III, Black, O., Jr., Maim, D. L., and Singh, M. (1977) Gastroenterology 73,1434-1449 3. Chandler, D. E. (1978) Life Sciences 23, 323-334 4. Selinger, Z., Eimerl, S., Savion, N., and Schramm, M. (1974) in Secretory Mechanisms of Exocrine Glands (Thord, N. A., and Petersen, 0. H., eds) pp. 68-87, Munksgaard, Copenhagen 5. Williams, J. A., and Lee, M. (1974) Biochem. Biophys. Res. Commun. 60,542-548 6. Case, R. M., and Clausen, T. (1973) J. PhysioZ., (Land.) 235,75102 7. Matthews, E. K., Petersen, 0. H., and Williams, J. A. (1973) J. Physiol. (Land.) 234,689-701 8. Gardner, J. D., ConIon, T. P., KIaeveman, H. L., Adams, T. D.,
by Pancreatic
Acini
7629
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