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Action of VIP and secretin on adenylate cyclase activity in acinar cells from guinea pig pancreas
GASTROENTEROLOGY
77:704-7X(
1979
Action of VIP and Secretin on Adenylate Cyclase Activity in Acinar Cells from Guinea Pig Pancreas EDWARD
1. OLINGER
and 1ERRY
D. GARDNER
The Section on Gastroenterology, Digestive Diseases Branch, National Institute of Arthritis, Metabolism and Digestive Disease, National Institutes of Health, Bethesda, Maryland
In membranes from dispersed acinar cells from guinea pig pancreas, adenylate cyclase activity was increased by vasoactive intestinal peptide, secretin, and 5’-guanylimidodiphosphate, a nucleotidase-resistant analogue of GTP. The time-course for stimulation of adenylate cyclase activity in membranes incubated with vasoactive intestinal peptide was rapid compared with that in membranes incubated with 5’-guanylimidodiphosphate. In membranes incubated with vasoactive intestinal peptide plus the nucleotide, the time-course for stimulation of enzyme activity was the same as that with the nucleotide alone. The guanyl nucleotide potentiated the increase in adenylate cyclase activity caused by secretin or vasoactive intestinal peptide in that the increase in enzyme activity caused by the nucleotide plus either of the peptides was significantly greater than the sum of the increase caused by each agent acting alone. Without the guanyl nucleotide the threshold concentration for stimulation of adenylate cyclase activity by vasoactive intestinal peptide or secretin was greater than z nM whereas with the nucleotide a significant increase in enzyme activity could be detected with concentrations of vasoactive intestinal peptide or secretin as low as 10 pM. The dose response curves for peptide-stimulated adenylate cyclase activity were monophasic without 5’guanylimidodiphosphate but were biphasic with the nucleotide. These actions of 5’-guanylimidodiphosphate were temperature-dependent, could not be reReceived January 17,1979. Accepted May 30,1979. Address requests for reprints to: Dr. Jerry D. Gardner, National Institutes of Health, Bldg. 10 Room 9D-15, Bethesda, Maryland 20205. Dr. Olinger’s present address is: Gastroenterology Section, Northwestern Memorial Hospital, 1420 Wesley Pavilion, 250 E. Superior St., Chicago, Illinois 60611. We thank Mary Ernst for preparing this manuscript for publication. 0 1979 by the American Gastroenterological Association OOlS-5085/79/100704-10$02.00
versed by washing, and did not reflect changes in membrane binding of the peptide or of the nucleotide. Furthermore, the effects of 5’-guanylimidodiphosphate could be inhibited by GTP or GDP, and once established, could be reversed by GTP or GDP. These results indicate that, in acinar cells from guinea pig pancreas, interactions of guanyl nucleotides with the GTP-regulatory site of adenylate cyclase may be an important determinant of the ceJJuJar response to vasoactive intestinal peptide and secretin.
In previous studies’-” we have found that acinar cells from guinea pig pancreas possess two distinct classes of receptors that mediate the action of VIP and secretin on cellular cyclic AMP. One class is VIP-preferring in that it has a high affinity for VIP and a low affinity for secretin, whereas the other class is secretin-preferring in that it has a high affinity for secretin and a low affinity for VIP. These two classes of receptors differ not only in their relative affinities for VIP and secretin, but also in their affinities for C-terminal fragments and analogues of secretin.‘-“,” In contrast to the biphasic dose-response curve for the action of VIP on cyclic AMP on intact acinar cells, the dose-response curve for the action of VIP in adenylate cyclase activity in broken acinar cells is monophasic.’ Furthermore, in membranes from pancreatic acinar cells, VIP is more potent than secretin in activating adenylate cyclase.’ GTP is known to enhance the stimulation of adenylate cyclase by a variety of hormones and neurotransmitters in many tissues (for review, see reference 8) including pancreas.‘.” Moreover, in some systems, GTP appears to be absolutely required for enzyme activation by a hormone.’ This action of GTP has been shown to reflect its interaction with a regulatory site that is distinct from the catalytic site of the enzyme.“~“-‘5 Following binding of the nucle-
0ctoh:r
1979
ADENYLATE CYCLASE IN PANCREATIC ACINAR CELLS
otide to the regulatory site, modification of enzyme activity occurs via a relatively slow, temperature-dependent process, the molecular basis of which has not yet been defined.H,“m’7 The kinetics of the action of GTP on hormone-stimulated adenylate cyclase are complex and influenced at least in part by degradation of the nucleotide.“.‘3.‘5~‘7 To attempt to simplify studies of the action of GTP, many investigators have used an analog of GTP, 5’guanylimidodiphosphate (GMPPNP) which is not degraded by nucleotidases.“~“‘.1s-‘8 Results from several different laboratories indicate that GMPPNP interacts with the GTP regulatory site and like the native nucleotide modifies enzyme activity by a slow, temperature-sensitive process which is subject to modification by a variety of different incubation conditions.” I,~1’~17As has been pointed out by others,‘“” adding GTP analogues directly to the adenylate cyclase assay can give rise to results which are difficult to interpret and, at times, misleading. Since the stimulation of adenylate cyclase caused by GMPPNP is slowly reversible, the complexities of the action of the nucleotide can be simplified by first incubating membranes with the nucleotide analogue and then washing the membranes before assay.“‘,” In the present study we have examined the actions of GMPPNP, VIP, and secretin on adenylate cyclasc activity in membranes from dispersed pancreatic acinar cells and have investigated the possibility that the difference between the dose-response curves for the actions of VIP and secretin on cyclic AMP in intact acinar cells and the pattern of action of the peptides on adenylate cyclase in broken acinar cells might reflect an action of guanyl nucleotides. We have found (a) that the pattern of action of GMPPNP on adenylate cyclase differs substantially from that of secretin and VIP, (b) that GMPPNP alters the configuration of the dose-response curves for the action of VIP and secretin on adenylate cyclase, and (c) that this action of the nucleotidc is not attributable to changes in binding of the peptide to its receptor.
Materials NIH strain guinea pigs (250-400 g) were obfrom the Small Animals Section, Veterinary Re-
Male
tained
sources Branch, National Institutes of Health (Bethesda, Md.) [(Y”‘JATP (lo-20 Ci/mmole) and cyclic [“HIAMP (22 Ci/mmole) from New England Nuclear Corp. (Boston, Mass.); [,‘H]GMPPNP (8.7Ci/mmole) from International Chemical and Nuclear Co. (Irvine, Calif.); carrier-free Na “‘1 (400-600 &i/pl) in 0.1 N NaOH from Radiochemical Center (Amersham, England); chloramine T from Eastman Kodak Co. (Rochester, N.Y.); sodium metabisulfite from Aldrich Chemical Co. (St. Louis, MO.); bacitracin from Calbiochem (La Jolla, Calif.); bovine serum albumin from
705
Miles Laboratories, Inc. (Kankakee, 111.); purified soybean trypsin inhibitor from Worthington Biochemical Corp. (Freehold, N.J.); and crude collagenase (Clostridium histolyticum, E.C. 3.4.4.19) and crude hyaluronidase (bovine testis, E.C. 3.2.1.35) from Sigma Chemical Co. (St. Louis MO.). Natural porcine secretin and porcine vasoactive intestinal peptide were gifts from Professor Viktor Mutt, Gastrointestinal Hormone Research Unit, Karolinska Institutet (Stockholm, Sweden). Synthetic porcine secrt:tiIl!, .:,, (SQ 19,301) was a gift from Dr. M. A. Ondetti. Squibb lnstitute for Medical Research (Princeton, N.J.). All other chemicals used were of the highest grade commercially available.
Methods Iodination
of VIP
VIP was radioiodinated with carrier-free ““I using a modification of the procedure of Hunter and Grcenwood’!’ described previously.’ The specific activity of ““lVIP, assuming complete recovery of the peptide, was 900 Ci/mmole.
Tissue
Preparation
Dispersed acinar cells from guinea pig pancreas were prepared using minor modifications of the procedure of Amsterdam and Jamieson’“~” reported previously..“‘,‘4 Partially purified plasma membranes from dispersed acinar cells were prepared as described previously’ and were resuspended in iced 50 mM TrisCl (pH 7.5), 1 mM di0.15% (wt/vol) albumin (“suspending mcthiothieitol, dium”), just before use.
Adenylate
CycJase
Activity
Membranes were suspended in 50 mM TrisCl (pH 7.5), 1 mM dithiothreitol and 0.15% (wt/vol) albumin irlcubated with various agents at 4’C or 37°C (first ucubation) and then washed by adding 50 vol of iced suspending medium. Membranes were sedimented by centrifugation at 1000g for 5 min, and the supernatant was aspirated. Membranes were resuspended to their original volume with suspending medium and incubated at the appropriate temperature (second incubation). At various times during the second incubation, aliquots of the incubation mixture were taken to measure adenylatc cyclase activity. Enzyme activity was determined by measuring the formation of cyclic [“‘PIAMP from [@P]ATP as described previously.7 Unless specified otherwise assays were for 6 min at 37’C and contained membranes (1.3-2.6 mg protein/ml), 50 mM TrisCl, pH 7.5, 1 mM dithiothrci10 mM KCl, 0.125 tol, 5 mM MgCl,, 9 mM theophylline. mM [a”‘P]ATP (1 &i), ATP regenerating system (14 pg creatine phosphokinase, 10 mM creatine phosphate), and 0.15% (wt/vol) albumin in a total volume of 75 ~1. The assay was initiated by adding membranes and was stopped by adding 500 ~1 of 5% (vol/vol) perchloric acid followed by 100 ~1 of a solution containing 2.5% (wt/vol)
OLINGER AND GARDNER
706
71
1
1
GASTROENTEROLOGY
14,
1
I
7-I
~~~ 1
Vol. 77, No. 4, Part 1
]
B 6;
12-
A
i
F
E
2
5
I
I
0
60 TIME
L
10
imlnutesl
sodium dodecylsulfate, 50 mM ATP, and 1.25 mM cyclic [“HIAMP (approximately 20,000 cpm). The reaction tube was placed at 4°C for 5 min. Five hundred microliters of 0.8 M Tris was added, and the mixture was centrifuged at 1000 g for 5 min. Isolation and detection of cyclic [“‘PIAMP in the supernatant was performed using the technique of Salomon et al.‘” as reported previously.‘,‘” All results were corrected for recovery of cyclic [3H]AMP, which was 55-70’70. Blank values for “P were 4-7 cpm above machine background. Under all conditions tested the rate of formation of cyclic [“‘PIAMP was constant for at least 12 min. Binding of rH]GMPPNP or ““I-VIP was measured by incubating membranes (1.346 mg protein/ml) in 750 pl of suspending medium containing lo-‘M [“HIGMPPNP or 5 x lo-“M ?-VIP. At specified times, duplicate 100 ~1 sammedium ples were layered over 300 ~1 of iced suspending in polyethelene microcentrifuge tubes. The membranes were washed three times by alternate centrifugation (10,000 g for 15 set in a Beckman model 153 microfuge, Spinco Div., Beckman Instruments, Inc., Palo Alto, Calif.) and resuspension. The washing procedure required 2 min. Membranes were then treated with 100 ~15% (vol/vol) Triton X-100 and transferred to a liquid scintillation vial containing 20 ml solution composed of 15 parts toluene, 5 parts Triton X-100, and 1 part Liquifluor (New England Nuclear Corp). At some time during the incubation, duplicate 100 ~1 samples were added directly to a scintillation
vial to measure total radioactivity in the sample. This value was used to calculate binding of [“HIGMPPNP or ““I-VIP as the percent of total radioactivity present in the sample. Nonsaturable binding was that binding measured in incubations containing lo-“M nonradioactive GMPPNP or 5 x lo-“M VIP. This value was always less than 10% of the binding observed in the absence of nonradioactive compound. All values for binding are expressed as saturable binding (total binding minus nonsaturable binding). Protein was determined using the method of Lowry et al.” with bovine serum albumin as a standard.
Results Binding
of rH]GMPPNP
and
““I-VIP
At 37°C saturable binding of [“HIGMPPNP to membranes from pancreatic acinar cells was moder-
Figure
1. Binding
of [“HI GMPPNP or ‘““I-VIP to membranes from pancreatic acinar cells. Membranes were suspended in 50 mM TrisCI (pH 7.5), 1 mM dithiothreitol. and
0.15% (wt/vol) albumin and incubated with lo-’ M [“HI GMPPNP (panel A) or 5 x lo-” M ““I-VIP (panel B) at the indicated temperature. Saturable binding of radioactivity was determined at the times indicated. In each experiment each value was determined in triplicate, and results shown are means from three separate experiments. Vertical bars represent 1 SD.
ately rapid and reached a steady state by 30 min (Figure 1A). Reducing the incubation temperature to 4’C reduced saturable binding of [“HIGMPPNP by more than 90% (Figure 1A). At 37°C saturable binding of ““I-VIP became maximal by 20 min and then decreased by approximately 10% during the subsequent 40 min (Figure 1B). Reducing the incubation temperature to 4°C reduced saturable binding of “‘IVIP to 25% of that seen at 37’C (Figure 1B). With membranes which had been preincubated with [“HIGMPPNP or ““I-VIP at 37’C for 20 min, washed, and resuspended, there was no dissociation of bound radioactivity during a 60-min incubation at 4°C (not shown). When membranes were incubated at 37°C instead of 4”C, bound ““I-VIP dissociated at a rate of 0.8% per minute while [“HIGMPPNP dissociated at a rate of 1.3% per minute (not shown). Vasoactive intestinal peptide did not alter binding of [“HIGMPPNP and GMPPNP did not alter binding of ““I-VIP. With membranes incubated at 37°C for 30 min saturable binding of [“HIGMPPNP in the presence of 1 1-1M VIP was 92 f 12% of that measured without VIP (mean f 1 SD of three experiments). Under identical conditions saturable binding of ‘““I-VIP in the presence of 0.1 mM GMPPNP was 105 f 9% of that measured without GMPPNP (mean + 1 SD of three experiments).
Effect of Jncubation Temperature on Basal and Stimulated Adenylate CycJase Activity To examine the effect of incubation temperature on adenylate cyclase activity, acinar cell membranes were incubated for 60 min at 4°C for 37°C with no additions (basal), VIP, or GMPPNP. Enzyme activity was assayed at different times during the incubation. When membranes were incubated at 4”C, basal adenylate cyclase activity remained constant for at least 60 min (Figure 2A). In contrast, at 37’C basal enzyme activity decreased by 70% within 15 min, by 65% after 45 min, and remained constant from 45 to 60 min (Figure 2A). When membranes
0ctobr:r 1979
ADENYLATE
CYCLASE
IN PANCREATIC
4OiL;
ACINAR
10
2'0 30
707
i 4:__;
A-_
i_~
CELLS
! 40
50
$0
TIME (minutes)
Figure
2. Effect of temperature on the increase in adenylate cyclase activity caused by VIP or GMPPNP. Membranes were suspended in 50 mM TrisCl (pH 7.5), 1 mM dithiothreitol, 0.15% (wt/vol) albumin and incubated with no additions (panel A), VIP (panel B), or GMPPNP (panel C) at the indicated temperature for 60 min. At various times during the incubation aliquots were taken to determine adenylate cyclase activity during a 6-min reaction at 37“C. In each experiment each value was determined in duplicate, and results shown are means from four separate experiments. Vertical bars represent 1 SD.
were incubated with lo-’ M VIP at 4”C, adenylate cyclase activity was approximately twice that observed in membranes incubated without VIP and remained constant for 60 min (Figure 2B). In membranes incubated with lo-’ M VIP at 37”C, enzyme activity decreased progressively to less than 10% of the initial value after 45 min (Figure 2B). When membranes were incubated with 10m4 M GMPPNP at 4”C, adenylate cyclase activity was approximately twice that observed in membranes incubated without GMPPNP and remained constant for 60 min (Figure 2C). In contrast to results with VIP, in membranes incubated with 10e4 M GMPPNP at 37”C, enzyme activity increased XI- to 20-fold by 15 min and remained constant thereafter (Figure 2C).
Effect of Removing Activity Measured Incubation
the Agonist on Enzyme During a Subsequent
When membranes were incubated with a particular agent at 4°C and then assayed for enzyme activity at 37”C, the observed activation of adenylate cyclase (Figure 2B and 2C) could have occurred during the incubation at 4’C or during the 6-min assay at 37°C. To examine this possibility membranes were first incubated with 10m7 M VIP for 10 min at 4”C, washed to remove free VIP, and then assayed for adenylate cyclase activity at various times during a second incubation at 4°C or 37’C (Figure 3A). Under these conditions stimulation of adenylate cyclase activity decreased progressively during a 60min second incubation at 37°C and reducing the temperature of the second incubation to 4’C significantly slowed the rate of decrease in VIP-stimulated
enzyme activity (Figure 3A). In membranes which had been first incubated with lo-“ M GMPPNP for 10 min at 4”C, washed, and then incubated at 4’C, GMPPNP-stimulated adenylate cyclase activity was similar in magnitude to that caused by lo-’ M VIP (cf. Figure 3B with Figure 3A) an9 did not change during the second incubation at 4°C (Figure 3B). When the temperature of the second incubation was 37°C instead of 4”C, GMPPNP-stimulated enzyme activity increased five-fold within 15 min and remained constant for the subsequent 30 min (Figure 3B). When membranes were first incubated with GMPPNP at 37°C and then washed, GMPPNP-stimulated adenylate cyclase activity was approximately 13-times greater than it was in membranes first incubated at 4”C, did not change during a second incubation at 4°C and increased slightly when the temperature of the second incubation was 37OC (Figure 3B).
Time-Course for Stimulation of Adenylate Cyclase Activity in Membranes First Incubated with VIP plus GMPPNP To test for a possible interaction bqtween the actions of VIP and GMPPNP on adenylite cyclase activity, membranes were first incubated with both agents for 10 min, washed, and then incubated at 37’C or 4°C (Figure 3C). With VIP plus GMPPNP the pattern of stimulation and of the effect of different incubation temperatures were similar to those seen with GMPPNP alone (cf. Figure 3C with Figure 3B). That is, stimulation of enzyme activity was greater in membranes first incubated at 37OC than it was in membranes first incubated at 4°C and the increase
708
OLINGER AND GARDNER
GASTROENTEROLOGY
in enzyme activity did not change during a second incubation at 4”C, but increased during the first 15 min of a second incubation at 37°C (Figure 3). With membranes first incubated at 4’C, the increase in enzyme activity caused by the two agents in combiprotein/l0 min) was signation (170 & 15 pmol/mg nificantly greater than the additive effects (VIP alone, 19 + 3 plus GMPPNP alone, 20 + 3 = 49 + 7) (mean + 1 SD from three experiments). In addition, in membranes first incubated with VIP plus GMPPNP at 4°C the increase in adenylate cyclase activity during a second incubation at 37’C (130 + 18 pmol/mg protein/l0 min) was significantly greater than that seen with membranes first incubated with GMPPNP alone (80 + 11) (mean f 1 SD from three experiments).
Reversibility Stimulated
of the Decrease in VIPAdenylate CycJase Activity
To examine the reversibility of the decrease in adenylate’cyclase activity in membranes first incubated with VIP, washed, and incubated at 37’C, GMPPNP or VIP was added after adenylate cyclase had reached a minimum value. In membranes first incubated with 10m7 M VIP at 4°C for 10 min, washed to remove free VIP, and then incubated at 37”C, adenylate cyclase activity decreased progressively during the second incubation and by 45 min reached the same value as that in membranes which had been first incubated without VIP (Figure 4). In membranes which had been first incubated with or 28
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A
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Vol. 77, No. 4, Part 1
without VIP, adding the peptide after 45 min of the second incubation caused a three-fold increase in adenylate cyclase activity by 60 min and this increase persisted for the remainder of the second incubation (Figure 4). Adding GMPPNP after 45 min of the second incubation also increased adenylate cyclase activity (Figure 4). However, in contrast to the effect of adding VIP, the increase in adenylate cyclase activity caused by adding GMPPNP to membranes which had been first incubated with VIP was 60% greater than that obtained with membranes which had been first incubated without VIP (Figure 4). Ability of GMPPNP to Modify VIP or Secretin on Adenylate Activity
the Action CycJase
of
To examine further the effects of GMPPNP on the activation of adenylate cyclase caused by VIP or secretin, membranes were incubated with varying concentrations of VIP or secretin with or without lo-“ M GMPPNP, washed, and assayed for adenylate cyclase activity (Figure 5). In membranes incubated with VIP alone a small but significant increase in adenylate cyclase activity could be detected with 3 X lOmy M VIP and maximal stimulation occurred incubated with with 3 x lo-’ M VIP. In membranes VIP plus GMPPNP, as the concentration of VIP increased, VIP-stimulated enzyme activity could be detected at 10-l’ M VIP, increased progressively to a plateau at 3 x lo-” M VIP, and then increased to a
1
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370 45
60
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15
30
45
so
TIME (minutes) Figure 3. Effect of temperature
on the increase in adenylate cyclase activity caused by VIP or GMPPNP. Membranes were suspended in 50 mM T&Cl (pH 7.5), 1 mM dithiothreitol, 0.15% (wt/vol) albumin, and incubated with no additions or the indicated agents for 10 min (first incubation). The membranes were then washed once with 56 vol of iced suspending medium. After centrifugation at 1000 g for 5 min and aspiration of the supernatant. membranes were resuspended to their original volume and incubated at 4’C or 37’C for 60 min (second incubation). At various times during the second incubation aliquots were taken to determine adenylate cyclase activity during a 6-min reaction at 37°C. Values given are for the increase in adenylate cyclase activity caused by the indicated agents and were calculated by subtracting enzyme activity measured at the same time using membranes which were processed identically, but contained no additions. Open symbols, first incubation at WC; closed symbols, first incubation at 4°C. Triangles, second incubation at 37°C; circles, second incubation at 4’C. In each experiment each value was determined
in duplicate, and results shown are means from three separate experimentk
Vertical bars represent 1 SD.
ADENYLATE
32
2 k
Lug
Ei
GMPPNP 1104 MI
4*
\
5 4c
pi
; 0
15
30
- -45
60
75
90
Figure 4. Time-course of adenylate cyclase activity in membranes from pancreatic acinar cells. Membranes were suspended in 50 mM TrisCl (pH 7.5). 1 mM dithiothreitol and 0.15% (wt/vol) albumin and incubated for 10 min at 4’C with or without 10e7 M VIP (first incubation). The membranes were washed once by adding 50 vol of iced suspending medium containing no VIP. After centrifugation at 1000 g for 5 min and aspiration of the supernatant, membranes were resuspended to their original volume with suspending medium and incubated at 37’C (second incubation). Adenylate cyclase activity was determined at different times during the second incubation, using a 6-min reaction at 37°C. After 45 min of the second incubation, VIP or CMPPNP was added, and the incubation was continued for 45 min. Open symbols. first incubation with VIP; closed symbols, first incubation without VIP. In each experiment each value was determined in duplicate, and results shown are means from three separate experiments. Vertical bars represent I SD.
2 %;lsl
240 220 200
240 220 210 200 180 160 140 120 100 80
A ,$,
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4
Y
I plus GMPPNP (10-Q Ml
IN PANCREATIC
To examine the ability of VIP to increase in adenylate cyclase activity GMPPNP, membranes were incubated ent concentrations of GMPPNP alone or VIP, washed, and assayed for adenylate
520 484 440 400 360 320 280 240 200
S
plus GMPPNP (104 M)
J
VIP (log M)
Figure
7og
; _.__;_~;--+%“ e -10
-9
-8
-7
SECRETIN (log M)
-6
-5
modify the caused by with differplus lo-’ M cyclase ac-
C
plus VIP (107 M) ‘
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CELLS
Effect of VIP on the Increase in Adenylate Cyclase Activity Caused by GMPPNP
?,
-5
ACINAR
second plateau as the VIP concentration increased from 3 x lo-” M to lo-’ M (Figure 5A). The maximal increase in adenylate cyclase activity caused by VIP was 10 times greater in membranes incubated with VIP plus GMPPNP than it was in membranes incubated with VIP alone (Figure 5A). Results with secretin were similar to those obtained with VIP. In membranes incubated with secretin alone a small, but significant increaSe in adenylate cyclase activity could be detected with lo-” M secretin and enzyme activity increased progressively as the secretin concentration was increased to 3 X lo-” M (Figure 5 B). In membranes incubated with secretin plus GMPPNP, as the concentration of secretin increased, stimulation of enzyme activity could be detected at lo-” M secretin, increased to a plateau at lo-“’ M secretin, remained constant until lo-” M secretin and at secretin-concentration above lo-” M there was a progressive increase in secretin-stimulated adenylate cyclase activity (Figure 5B). As occurred with VIP, the maximal increase in adenylate cyclase activity caused by secretin was 10 times greater in membranes incubated with secretin plus GMPPNP than it was in membranes incubated with secretin alone (Figure 5B).
36
3
CYCLASE
120 80 40 0
i
I
Alone
-6
5
_ :’ -8
-7
4
3
GMPPNP (log M1
5. Effect of VIP, secretin, and GMPPNP on adenylate cyclase activity in membranes from pancreatic acinar cells. Membranes were suspended in 50 mM TrisCl (pH 7.5). 1 mM dithiotheitol and 0.15% (wt/vol) albumin, incubated with the indicated agents for 20 min and then assayed for adenylate cyclase activity during a 6-min reaction at 37°C. Incubations containing GMPPNP were at 37°C whereas incubations without GMPPNP were at 4’C. Values given are for the increase in adenylate cyclase activity caused by the indicated concentration of the particular agent. For example, in panels A and B basal activity (19.6) was subtracted from all values determined with VIP or secretin alone, while activity with GMPPNP alone (297) was subtracted from all values determined with GMPPNP plus VIP or secretin. In each experiment each value was determined in duplicate and results shown are means from three separate experiments. Vertical bars represent 1 SD.
710
OLINGER
Table
1.
AND
GARDNER
GASTROENTEROLOGY
Effect of Secretin5_27 on Adenylase Cyclase Activity in Membranes from Pancreatic Acinar
Effect of Secretin5_z, Activity
Cells Adenylate
cyclase (pmoles cAMP/mg protein/l0 min)
Addition SecretinS-,, (30 PM) VIP (0.1 PM) VIP + Secretin,_,, GMPPNP (0.1 mM) GMj’PNP + VIP GMPPNP + Secretin,.,,
0.1 19.6 2.5 297.0 480.4
+ + f + +
0.2 1.6 0.2 26.7 32.0
271.3 + 18.0
Membranes were suspended in 50 mM TrisCl (pH 7.5), 1 mM dithiothreitol, and 0.15% (wt/vol) albumin, incubated with the indicated agents for 20 min and then assayed for adenylate cyclase activity using a 6-min reaction at 37°C. Assays containing GMPPNP were incubated at 37°C; others were at 4°C. Values given are for the increase in adenylate cyclase activity caused by the indicated agents and were calculated by subtracting enzyme activity measured in incubations which were processed identically, but contained no additions. In each experiment each value was determined in triplicate, and results given are means (fl SD) from four separate experiments.
tivity (Figure 5C). In membranes incubated without VIP, GMPPNP-stimulated enzyme activity could be detected at 3 X lo-’ M GMPPNP and was maximal at 1O-4 M GMPPNP (Figure 5C). In membranes incubated with VIP plus GMPPNP the configuration of the dose-response curve for enzyme activity stimulated by GMPPNP was unaltered, but the increase in enzyme activity caused by a maximally effective concentration of GMPPNP was increased by 60-70% (Figure 5C). Table
2. Effect of Guanyl Cyclase Acinar
Activity Cells
Nucleotides in Membranes
Vol. 77, No. 4, Part 1
on Adenylate
Cyclase
Secretin .5_-27, a C-terminal fragment of secretin which is a competitive antagonist of the action of secretin or VIP on adenylate cyclase activity in pancreatic acinar cells,‘~“~’ did not alter basal adenylate cyclase activity or the stimulation of enzyme activity caused by GMPPNP, but significantly inhibited the increase in cyclase activity caused by VIP (Table 1). Effects Action
of Different Guanyl of GMPPNP
Nucleotides
on
the
The increase in adenylate cyclase activity caused by GMPPNP alone or in the presence of VIP could be inhibited by guanyl nucleotides such as GTP, GDP, and GMP (Table 2) but not by guanosine or guanine (not shown). Not only were these nucleotides able to inhibit the increase in enzyme activity caused by GMPPNP, but they could also reverse the action of GMPPNP on adenylate cyclase activity (Figure 6). In membranes which had been first incubated at 37°C with GMPPNP or GMPPNP plus VIP, washed, and then incubated at 37’C, adding 5 mM GTP or 5 mM GDP during the second incubation caused progressive decrease in enzyme activity (Figure 6).
on Adenylate from Pancreatic
Adenylate cyclase (percent of control)
(10 ILM)
GMPPNP (10 pM) plus VIP (0.1 PM)
100 14 + 2 15 r+ 2 59 + 6
100 16 +2 23 + 3 72 f 4
GMPPNP Addition None GTP (5 mM) GDP (5 mM) GMP (5 mM)
Membranes were suspended in 50 mM TrisCl (pH 7.5). 1 mM dithiothreitol, 0.15% (wt/vol) albumin. To determine adenylate cyclase activity, membranes were incubated for 10 min at 37°C with the indicated agents and washed once by adding 50 vol of iced, suspending medium. After centrifugation at 1000 g for 5 min and aspiration of the supcrnatant, membranes were resuspended to their original volume and assayed for adenylate cyclase activity during a 6-min reaction at 37°C. Values are expressed as percent of that determined with no additions (i.e., with GMPPNP alone or with GMPPNP plus VIP alone). In each experiment each value was determined in duplicate, and results given are means (il SD) from three separate experiments.
Effect of guanyl nucleotides on the activation of adenylate cyclase caused by GMPPNP. Membranes were suspended in 50 mM TrisCl (pH 7.5), 1 mM dithiothreitol and 0.15% (wt/vol) albumin and incubated with GMPPNP or GMPPNP plus VIP for 10 min at 37°C (first incubation). The membranes were then washed by adding 50 vol of iced suspending medium containing no VIP or GMPPNP. After centrifugation at 1000 g for 5 min and aspiration of the supernatant, membranes were resuspended to their original volume with iced suspending medium containing no additions, GDP, or GTP and incubated for 60 min at 37“C (second incubation). At different times during the second incubation samples were taken to determine adenylate cyclase activity using a 6-min reaction at 37°C. In each experiment each value was determined in duplicate, and results shown are means from three separate experiments. Vertical bars represent 1 SD.
0c:tolwr 1979
ADENYLATE CYCLASE IN PANCREATIC ACINAR CELLS
Discussion In agreement with previous studies in other tissUes7.~1 II)ZH:,(I)the present results illustrate that although GMPPNP, secretin, and VIP can activate adenylate cyclase in membranes from pancreatic acinar cells, the characteristics of activation by the guanyl nucleotide differ substantially from those of the activation caused by the peptides. At 37”C, basal as well as VIP-stimulated enzyme activity decreased progressively to a minimum value by 30-45 min whereas GMPPNP-stimulated activity increased and became maximal after 15 min. Reducing the incubation temperature to 4°C abolished the time-dependent decrease in basal and VIP-stimulated activity. From the present studies, we cannot determine whether the time-dependent decrease in basal and VIP-stimulated adenylate cyclase activity observed at 37°C represented loss of some membrane-associatccl cofactor required for enzyme activity or deterioration (i.e., irreversible inactivation) of the membrane preparation. Our finding that after 45 min of incubation enzyme activity could be partially stimulated by adding VIP or GMPPNP, indicates that at least part of the decrease in enzyme activity may have resulted from loss of one or more hypothetical cofactors which are required to support basal as well as stimulated enzyme activity. In agreement with findings in other tissues,“.‘“.‘n-‘” the relatively slow, temperature-dependent activation of adenylate cyclasc in acinar cell membranes incubated with GMPPNP appeared to reflect changes which occurred after nucleotide binding since membranes incubated with GMPPNP and washed to remove free nuclcotidc also showed an increase in adenylate cyclase activity during the first 15 min during a second incubation at 37’C. The ability of GMPPNP to stimulate adenylate cyclase activity and to modify the stimulation caused by VIP or secretin appears to reflect its interaction with the GTP regulatory site since, as has been observed in other systcms,“.‘R.“~‘” the action of the nucleotide analoguc could be inhibited by GTP as well as by GDP and GMP. In some tissues GMPPNP has been found to cause a persistent activation of adenylate cyclase which is not reversible by washing the membranes to remove the free nucleotide or by adding guanyl nucleotides such as GTP or GDP.“.“‘~‘“~‘”In the present studies we found that the action of GMPPNP could not be reversed by washing the membranes, but could be reversed by adding GTP or GDP. These findings illustrate that in membranes from pancreatic acinar cells, guanyl nucleotides which are susceptible to hydrolysis can reverse the persistent activation caused by GMPPNP. Because the molecular basis of action of guanyl nucleotides
711
on adenylate cyclase is not known, it seems unreasonable to speculate about the possible mechanisms by which native guanyl nucleotides are able to reverse the activity of adenylate cyclase caused by GMPPNP. Our findings do indicate that in membranes from pancreatic acinar cells, the association of guanyl nucleotides with the GTP regulatory site do not reflect a simple bimolecular reaction. In agreement with the findings of Svoboda et al.!‘,“’ GMPPNP potentiated the action of secretin and VIP on adenylate cyclase activity in membranes from pancreatic acinar cells, i.e., the increase in adenylate cyclase activity caused by GMPPNP plus VIP or secretin was substantially greater than the sum of the increase caused by each agent acting alone. This potentiation was not attributable to changes in binding of GMPPNP to the GTP regulatory site or of VIP to its receptors since neither agent caused detectable changes in binding of the other agents. In some systems where guanyl nucleotides enhance the activation of adenylate cyclase caused by a hormone or neurotransmitter, the nucleotide has been found to accelerate the rate of dissociation of the bound agonist and by so doing, reduce the amount of agonist bound at the steady state.“,“” Potentiation of the stimulation of adenylate cyclase caused by VIP plus GMPPNP required that the VIP receptor be occupied by an active agonist since potentiation of adenylate cyclase activity did not occur with the guanyl nuclcotide plus secretin, 27 which binds to the hormone of adenyreceptors,‘~“~7 but does not cause activation late cyclase. Finally, potentiation did not require that VIP and GMPPNP be added to the incubation simultaneously. In membranes first incubated with VIP for 10 min at 4”C, washed, and then incubated at 37°C for 45 min, the increase in adenylate cyclase activity caused by adding GMPPNP to the second incubation was substantially greater than that in membranes first incubated without VIP, but otherwise treated identically. In addition to potentiating the action of agents on adenylate cyclase in membranes from pancreas”’ as well as virtually every other tissue studied, guanyl nucleotides can also alter the time-course of activation caused by various agonists (for review, see references 8,15,16,18). When guanyl nucleotides and hormones or neurotransmitters are added to the adenylate cyclase assay medium, several minutes are required to achieve maximal activation by the nucleotide while activation by hormones or neurotransmitters is maximal at the earliest time it is technically possible to take a sample.““‘~‘“~“’ The timecourse of activation of cyclase when a guanyl nucleotide plus a hormone or neurotransmitter is added to the cyclase assay medium is the same as that caused by adding the hormone or neurotransmitter alone. In
712
OLINGER
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the present studies the time-course of stimulation of adenylate cyclase by VIP plus GMPPNP was the same as that for the nucleotide alone. This similarity occurred in membranes incubated with both agents as well as in membranes incubated with both agents and then washed before a second incubation and was not altered by changing the incubation temperature. The basis for this difference is not known; however, the present findings as well as results of others illustrate that with or without an agonist, full stimulation of adenylate cyclase by GMPPNP requires at least 15 min of incubation at 37°C. In the present studies, GMPPNP not only potentiated the action of secretin and VIP and modified the time-course of action of the peptides, but also substantially altered the dose-response curves for the action of VIP and secretin. GMPPNP increased the sensitivity to VIP and secretin in that without the nucleotide, peptide concentrations of greater than 1 nM were required to cause detectable stimulation of adenylate cyclase activity, whereas with the nucleotide, significant stimulation of enzyme activity could be detected with peptide concentrations as low as 10 pM. GMPPNP also altered the configuration of the dose-response curves for VIP and secretin; however, VIP did not alter the configuration of the dose-response curve for the action of the guanyl nucleotide. Without GMPPNP, the dose-response curve for the action of VIP or secretin was monophasic whereas with the nucleotide, the doseresponse curves for the peptides were biphasic. In most tissues studied to date guanyl nucleotides have potentiated the increase in adenylate cyclase activity caused by hormones and neurotransmitters, but have not altered the sensitivity to stimulation by an agonist or the configuration of the dose-response curve.“.“-“’ In some systems, however, guanyl nucleotides have been shown to increase the sensitivity of adenylate cyclase to stimulation by various agonists.“~“~‘” Svoboda et al.“’ studying plasma membranes from rat pancreas found that guanyl nucleotidcs potentiated the increase in adenylate cyclase activity caused by peptides, caused a relatively small (approximately 50%) decrease in peptide concentration required to cause half-maximal enzyme activity, but did not alter the configuration of the dose-response curves for the actions of the peptides. The basis for the difference between the results obtained by Svoboda et al.“’ and those in the present studies are probably attributable to the different incubation conditions. The present results indicate that in intact acinar cells the interaction of guanyl nucleotides with the GTP-regulatory site may be an important determinant of the cellular response to VIP and secretin and suggest that GTP is required for optimal expression of the biologic activity initi-
GASTROENTEROLOGY
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Vol. 77, No. 4, Part 1
the secretin-prereceptors.
References 1. Christophe
JP, Cordon TP, Gardner JD: Interaction of porcine vasoactive intestinal peptide with dispersed pancreatic acinar cells from the guinea pig. Binding of radioiodinated peptide. J Biol Chem 251:4629-4634,1976 2. Robberecht P, Conlon TP, Gardner JD: Interaction of porcine vasoactive intestinal peptide with dispersed acinar cells from the guinea pig: structural requirements for effects of VIP and secretin on cellular cyclic AMP. J Biol Chem 251:4635-4639, 1976 3. Gardner JD, Conlon TP, Fink ML, et al: Interactions of peptides related to secretin with hormone receptors on pancreatic acinar cells. Gastroenterology 71:965-970, 1976 4. Gardner JD, Conlon TP, Beyerman HC, et al: Interaction of synthetic lo-tyrosyl analogs of secretin with hormone receptors on pancreatic acinar cells. Gastroenterology 73:52-56, 1977 JD, Jackson MJ: Regulation of amylase release from 5. Gardner dispersed pancreatic acinar cells. J Physiol270:439-454, 1977 JD, Rottman AJ, Natarajan S, et al: Interaction of se6. Gardner cretm,_,, and its analogues with hormone receptors on pancreatic acini. Biochem Biophys Acta 583:491-503,1979 JD: Effects of cholecystokinin on adeny7. Long, BW, Gardner late cyclase activity in dispersed pancreatic acinar cells. Gastroenterology 73:1008-1014.1977 Y, et al: Role of adenine and 8. Rodbell M, Lin MC, Salomon guaninc nucleotidcs in the activity and response of adenylate cyclase systems to hormones: evidence for multisite transition states. Adv Cyclic Nucl Res 5:3-29,1975 M, Robberecht P, Camus J, et al: Subcellular distri9. Svoboda bution and response to gastrointestinal hormones of adenylate cyclase in rat pancreas. Eur J Biochem 69:185-193.1976 P, Camus J, et al: Association of bind10. Svoboda M, Robbcrecht ing sites for guanine nucleotides with adenylate cyclase activation in rat pancreatic plasma membranes. Eur J Biochem 83:287-297,1978 11. Rodbell M, Birnbaumer L, Pohl SL, et al: The glucagon-sensitive adenyl cyclase system in plasma membranes of rat liver. An obligatory role of guanyl nucleotides in glucagon action. J Biol Chem 246:1877-1882,197l EJM: Activation of pigeon erythrocyte 12. Pfeuffer T, Helmreich membrane adenylate cyclase by guanyl nucleotide analogues and separation of a nucleotide binding protein. J Biol Chem 250:867-876,1975 P, Bennett V, Jacob S: Irreversible stimulation of 13. Cuatrecasas adenylate cyclase activity of fat cell membranes by phosphoramidate and phosphonatc analogs of GTP. J Membr Biol 23:249-278,1975 14. Lefkowitz R: Guanosine triphosphate binding sites in solubilized myocardium-relation to adenylatc cyclase activity. J Biol Chcm 250:1006-loll,1975 M: The catecholamine-responsive adenylate cy15. Schramm clase system and its modification by Sguanylimidodiphosphate. Adv Cyclic Nucl Res 5:105-115. 1975 GD, Speigel AM, Gardner JD: P-adrenergic recep16. Aurbach tors, cyclic AMP, and ion transport in the avian erythrocyte. Adv Cyclic Nucl Res 5:117-132,1975 P, Jacobs S, Bennett V: Activation of adcnylatc cy17. Cuatrecsas clase by phosphoramidate and phosphonate analogs of GTP: possible role of covalent enzyme-substrate intermediates in the mechanism of hormonal activation. Proc Nat1 Acad Sci USA 721739-1743.1975
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ADENYLATE
18. Londos C, Salomon Y, Lin MC, et al: 5’.guanylimidodiphosphate. a potent activator of adenylate cyclase systems in eukaryotic cells. Proc Nat1 Acad Sci USA 71:3087-3090, 1974 19. Hunter WM, Greenwood FC: Preparation of iodine-131 lahelled human growth hormone of high specific activity. Nature (Lend) 194:495-496.1962 20. Amsterdam A, Jamieson JD: Structural and functional characterization of isolated pancreatic exocrine cells. Proc Nat1 Acad Sci USA 69:3028-30321972 21. Amsterdam A, Jamieson JD: Studies on dispersed pancreatic cxocrine cells. I. Dissociation technique and morphologic characteristics of separated cells. J Cell Biol 63:1037-1056, 1974 22. Amsterdam A, Jamicson JD: Studies on dispersed pancreatic cxocrine cells. II. Functional characteristics of separated cells. J Cell Biol 63:1057-1073, 1974 23. Gardner JD, Conlon TP, Klaevcman HL, et al: Action of cholccystokinin and cholinergic agents on calcium transport in isolated pancreatic acinar cells. J Clin Invest 56:366-375, 1975 24. Gardner JO, Conlon TP, Adams TD: Cyclic AMP in pancre-
25. 26.
27.
28.
29.
30.
CYCLASE
IN PANCREATIC
ACINAR
CELLS
713
atic acinar cells: effects of gastrointestinal hormones. Gastroenterology 70:29-35, 1976 Salomon Y, Londos C, Rodhell M: A highly sensitive adenylate cyclase assay. Anal Biochcm 58:541-548,1974 Klaeveman HL. Conlon TP. Levy AG, et al: Effect of gastrointestinal hormones on adenylate cyclase activity in human jejunal mucosa. Gastroentcrology 68:667-675.1975 Lowry OH, Rosenhrough NJ, Farr AL, et al: Protein measurement with the Folin phenol reagent. J Biol Chem 193265-275, 1951 Rutten WJ, De Pant JJHHM, Bonting SL: Adenylate cyclast: in the rate pancreas. Properties and stimulation by hormones. Biochem Biuphys Acta 274:201-213,1972 Kempen HJM, DC Pont JJHHM. Bonting SL: Rat pancreas adenylate cyclase. 11. Inactivation and protection of its hormone receptor sites. Biochem Biophys Acta 370:573-584, 1974 Milutinovic S, Schulz I, Rosselin G: The interactions of secretin with pancreatic membranes. Biochem Biophys Acta 436:113-127. 1976
77:704-7X(
1979
Action of VIP and Secretin on Adenylate Cyclase Activity in Acinar Cells from Guinea Pig Pancreas EDWARD
1. OLINGER
and 1ERRY
D. GARDNER
The Section on Gastroenterology, Digestive Diseases Branch, National Institute of Arthritis, Metabolism and Digestive Disease, National Institutes of Health, Bethesda, Maryland
In membranes from dispersed acinar cells from guinea pig pancreas, adenylate cyclase activity was increased by vasoactive intestinal peptide, secretin, and 5’-guanylimidodiphosphate, a nucleotidase-resistant analogue of GTP. The time-course for stimulation of adenylate cyclase activity in membranes incubated with vasoactive intestinal peptide was rapid compared with that in membranes incubated with 5’-guanylimidodiphosphate. In membranes incubated with vasoactive intestinal peptide plus the nucleotide, the time-course for stimulation of enzyme activity was the same as that with the nucleotide alone. The guanyl nucleotide potentiated the increase in adenylate cyclase activity caused by secretin or vasoactive intestinal peptide in that the increase in enzyme activity caused by the nucleotide plus either of the peptides was significantly greater than the sum of the increase caused by each agent acting alone. Without the guanyl nucleotide the threshold concentration for stimulation of adenylate cyclase activity by vasoactive intestinal peptide or secretin was greater than z nM whereas with the nucleotide a significant increase in enzyme activity could be detected with concentrations of vasoactive intestinal peptide or secretin as low as 10 pM. The dose response curves for peptide-stimulated adenylate cyclase activity were monophasic without 5’guanylimidodiphosphate but were biphasic with the nucleotide. These actions of 5’-guanylimidodiphosphate were temperature-dependent, could not be reReceived January 17,1979. Accepted May 30,1979. Address requests for reprints to: Dr. Jerry D. Gardner, National Institutes of Health, Bldg. 10 Room 9D-15, Bethesda, Maryland 20205. Dr. Olinger’s present address is: Gastroenterology Section, Northwestern Memorial Hospital, 1420 Wesley Pavilion, 250 E. Superior St., Chicago, Illinois 60611. We thank Mary Ernst for preparing this manuscript for publication. 0 1979 by the American Gastroenterological Association OOlS-5085/79/100704-10$02.00
versed by washing, and did not reflect changes in membrane binding of the peptide or of the nucleotide. Furthermore, the effects of 5’-guanylimidodiphosphate could be inhibited by GTP or GDP, and once established, could be reversed by GTP or GDP. These results indicate that, in acinar cells from guinea pig pancreas, interactions of guanyl nucleotides with the GTP-regulatory site of adenylate cyclase may be an important determinant of the ceJJuJar response to vasoactive intestinal peptide and secretin.
In previous studies’-” we have found that acinar cells from guinea pig pancreas possess two distinct classes of receptors that mediate the action of VIP and secretin on cellular cyclic AMP. One class is VIP-preferring in that it has a high affinity for VIP and a low affinity for secretin, whereas the other class is secretin-preferring in that it has a high affinity for secretin and a low affinity for VIP. These two classes of receptors differ not only in their relative affinities for VIP and secretin, but also in their affinities for C-terminal fragments and analogues of secretin.‘-“,” In contrast to the biphasic dose-response curve for the action of VIP on cyclic AMP on intact acinar cells, the dose-response curve for the action of VIP in adenylate cyclase activity in broken acinar cells is monophasic.’ Furthermore, in membranes from pancreatic acinar cells, VIP is more potent than secretin in activating adenylate cyclase.’ GTP is known to enhance the stimulation of adenylate cyclase by a variety of hormones and neurotransmitters in many tissues (for review, see reference 8) including pancreas.‘.” Moreover, in some systems, GTP appears to be absolutely required for enzyme activation by a hormone.’ This action of GTP has been shown to reflect its interaction with a regulatory site that is distinct from the catalytic site of the enzyme.“~“-‘5 Following binding of the nucle-
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ADENYLATE CYCLASE IN PANCREATIC ACINAR CELLS
otide to the regulatory site, modification of enzyme activity occurs via a relatively slow, temperature-dependent process, the molecular basis of which has not yet been defined.H,“m’7 The kinetics of the action of GTP on hormone-stimulated adenylate cyclase are complex and influenced at least in part by degradation of the nucleotide.“.‘3.‘5~‘7 To attempt to simplify studies of the action of GTP, many investigators have used an analog of GTP, 5’guanylimidodiphosphate (GMPPNP) which is not degraded by nucleotidases.“~“‘.1s-‘8 Results from several different laboratories indicate that GMPPNP interacts with the GTP regulatory site and like the native nucleotide modifies enzyme activity by a slow, temperature-sensitive process which is subject to modification by a variety of different incubation conditions.” I,~1’~17As has been pointed out by others,‘“” adding GTP analogues directly to the adenylate cyclase assay can give rise to results which are difficult to interpret and, at times, misleading. Since the stimulation of adenylate cyclase caused by GMPPNP is slowly reversible, the complexities of the action of the nucleotide can be simplified by first incubating membranes with the nucleotide analogue and then washing the membranes before assay.“‘,” In the present study we have examined the actions of GMPPNP, VIP, and secretin on adenylate cyclasc activity in membranes from dispersed pancreatic acinar cells and have investigated the possibility that the difference between the dose-response curves for the actions of VIP and secretin on cyclic AMP in intact acinar cells and the pattern of action of the peptides on adenylate cyclase in broken acinar cells might reflect an action of guanyl nucleotides. We have found (a) that the pattern of action of GMPPNP on adenylate cyclase differs substantially from that of secretin and VIP, (b) that GMPPNP alters the configuration of the dose-response curves for the action of VIP and secretin on adenylate cyclase, and (c) that this action of the nucleotidc is not attributable to changes in binding of the peptide to its receptor.
Materials NIH strain guinea pigs (250-400 g) were obfrom the Small Animals Section, Veterinary Re-
Male
tained
sources Branch, National Institutes of Health (Bethesda, Md.) [(Y”‘JATP (lo-20 Ci/mmole) and cyclic [“HIAMP (22 Ci/mmole) from New England Nuclear Corp. (Boston, Mass.); [,‘H]GMPPNP (8.7Ci/mmole) from International Chemical and Nuclear Co. (Irvine, Calif.); carrier-free Na “‘1 (400-600 &i/pl) in 0.1 N NaOH from Radiochemical Center (Amersham, England); chloramine T from Eastman Kodak Co. (Rochester, N.Y.); sodium metabisulfite from Aldrich Chemical Co. (St. Louis, MO.); bacitracin from Calbiochem (La Jolla, Calif.); bovine serum albumin from
705
Miles Laboratories, Inc. (Kankakee, 111.); purified soybean trypsin inhibitor from Worthington Biochemical Corp. (Freehold, N.J.); and crude collagenase (Clostridium histolyticum, E.C. 3.4.4.19) and crude hyaluronidase (bovine testis, E.C. 3.2.1.35) from Sigma Chemical Co. (St. Louis MO.). Natural porcine secretin and porcine vasoactive intestinal peptide were gifts from Professor Viktor Mutt, Gastrointestinal Hormone Research Unit, Karolinska Institutet (Stockholm, Sweden). Synthetic porcine secrt:tiIl!, .:,, (SQ 19,301) was a gift from Dr. M. A. Ondetti. Squibb lnstitute for Medical Research (Princeton, N.J.). All other chemicals used were of the highest grade commercially available.
Methods Iodination
of VIP
VIP was radioiodinated with carrier-free ““I using a modification of the procedure of Hunter and Grcenwood’!’ described previously.’ The specific activity of ““lVIP, assuming complete recovery of the peptide, was 900 Ci/mmole.
Tissue
Preparation
Dispersed acinar cells from guinea pig pancreas were prepared using minor modifications of the procedure of Amsterdam and Jamieson’“~” reported previously..“‘,‘4 Partially purified plasma membranes from dispersed acinar cells were prepared as described previously’ and were resuspended in iced 50 mM TrisCl (pH 7.5), 1 mM di0.15% (wt/vol) albumin (“suspending mcthiothieitol, dium”), just before use.
Adenylate
CycJase
Activity
Membranes were suspended in 50 mM TrisCl (pH 7.5), 1 mM dithiothreitol and 0.15% (wt/vol) albumin irlcubated with various agents at 4’C or 37°C (first ucubation) and then washed by adding 50 vol of iced suspending medium. Membranes were sedimented by centrifugation at 1000g for 5 min, and the supernatant was aspirated. Membranes were resuspended to their original volume with suspending medium and incubated at the appropriate temperature (second incubation). At various times during the second incubation, aliquots of the incubation mixture were taken to measure adenylatc cyclase activity. Enzyme activity was determined by measuring the formation of cyclic [“‘PIAMP from [@P]ATP as described previously.7 Unless specified otherwise assays were for 6 min at 37’C and contained membranes (1.3-2.6 mg protein/ml), 50 mM TrisCl, pH 7.5, 1 mM dithiothrci10 mM KCl, 0.125 tol, 5 mM MgCl,, 9 mM theophylline. mM [a”‘P]ATP (1 &i), ATP regenerating system (14 pg creatine phosphokinase, 10 mM creatine phosphate), and 0.15% (wt/vol) albumin in a total volume of 75 ~1. The assay was initiated by adding membranes and was stopped by adding 500 ~1 of 5% (vol/vol) perchloric acid followed by 100 ~1 of a solution containing 2.5% (wt/vol)
OLINGER AND GARDNER
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]
B 6;
12-
A
i
F
E
2
5
I
I
0
60 TIME
L
10
imlnutesl
sodium dodecylsulfate, 50 mM ATP, and 1.25 mM cyclic [“HIAMP (approximately 20,000 cpm). The reaction tube was placed at 4°C for 5 min. Five hundred microliters of 0.8 M Tris was added, and the mixture was centrifuged at 1000 g for 5 min. Isolation and detection of cyclic [“‘PIAMP in the supernatant was performed using the technique of Salomon et al.‘” as reported previously.‘,‘” All results were corrected for recovery of cyclic [3H]AMP, which was 55-70’70. Blank values for “P were 4-7 cpm above machine background. Under all conditions tested the rate of formation of cyclic [“‘PIAMP was constant for at least 12 min. Binding of rH]GMPPNP or ““I-VIP was measured by incubating membranes (1.346 mg protein/ml) in 750 pl of suspending medium containing lo-‘M [“HIGMPPNP or 5 x lo-“M ?-VIP. At specified times, duplicate 100 ~1 sammedium ples were layered over 300 ~1 of iced suspending in polyethelene microcentrifuge tubes. The membranes were washed three times by alternate centrifugation (10,000 g for 15 set in a Beckman model 153 microfuge, Spinco Div., Beckman Instruments, Inc., Palo Alto, Calif.) and resuspension. The washing procedure required 2 min. Membranes were then treated with 100 ~15% (vol/vol) Triton X-100 and transferred to a liquid scintillation vial containing 20 ml solution composed of 15 parts toluene, 5 parts Triton X-100, and 1 part Liquifluor (New England Nuclear Corp). At some time during the incubation, duplicate 100 ~1 samples were added directly to a scintillation
vial to measure total radioactivity in the sample. This value was used to calculate binding of [“HIGMPPNP or ““I-VIP as the percent of total radioactivity present in the sample. Nonsaturable binding was that binding measured in incubations containing lo-“M nonradioactive GMPPNP or 5 x lo-“M VIP. This value was always less than 10% of the binding observed in the absence of nonradioactive compound. All values for binding are expressed as saturable binding (total binding minus nonsaturable binding). Protein was determined using the method of Lowry et al.” with bovine serum albumin as a standard.
Results Binding
of rH]GMPPNP
and
““I-VIP
At 37°C saturable binding of [“HIGMPPNP to membranes from pancreatic acinar cells was moder-
Figure
1. Binding
of [“HI GMPPNP or ‘““I-VIP to membranes from pancreatic acinar cells. Membranes were suspended in 50 mM TrisCI (pH 7.5), 1 mM dithiothreitol. and
0.15% (wt/vol) albumin and incubated with lo-’ M [“HI GMPPNP (panel A) or 5 x lo-” M ““I-VIP (panel B) at the indicated temperature. Saturable binding of radioactivity was determined at the times indicated. In each experiment each value was determined in triplicate, and results shown are means from three separate experiments. Vertical bars represent 1 SD.
ately rapid and reached a steady state by 30 min (Figure 1A). Reducing the incubation temperature to 4’C reduced saturable binding of [“HIGMPPNP by more than 90% (Figure 1A). At 37°C saturable binding of ““I-VIP became maximal by 20 min and then decreased by approximately 10% during the subsequent 40 min (Figure 1B). Reducing the incubation temperature to 4°C reduced saturable binding of “‘IVIP to 25% of that seen at 37’C (Figure 1B). With membranes which had been preincubated with [“HIGMPPNP or ““I-VIP at 37’C for 20 min, washed, and resuspended, there was no dissociation of bound radioactivity during a 60-min incubation at 4°C (not shown). When membranes were incubated at 37°C instead of 4”C, bound ““I-VIP dissociated at a rate of 0.8% per minute while [“HIGMPPNP dissociated at a rate of 1.3% per minute (not shown). Vasoactive intestinal peptide did not alter binding of [“HIGMPPNP and GMPPNP did not alter binding of ““I-VIP. With membranes incubated at 37°C for 30 min saturable binding of [“HIGMPPNP in the presence of 1 1-1M VIP was 92 f 12% of that measured without VIP (mean f 1 SD of three experiments). Under identical conditions saturable binding of ‘““I-VIP in the presence of 0.1 mM GMPPNP was 105 f 9% of that measured without GMPPNP (mean + 1 SD of three experiments).
Effect of Jncubation Temperature on Basal and Stimulated Adenylate CycJase Activity To examine the effect of incubation temperature on adenylate cyclase activity, acinar cell membranes were incubated for 60 min at 4°C for 37°C with no additions (basal), VIP, or GMPPNP. Enzyme activity was assayed at different times during the incubation. When membranes were incubated at 4”C, basal adenylate cyclase activity remained constant for at least 60 min (Figure 2A). In contrast, at 37’C basal enzyme activity decreased by 70% within 15 min, by 65% after 45 min, and remained constant from 45 to 60 min (Figure 2A). When membranes
0ctobr:r 1979
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4OiL;
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2'0 30
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! 40
50
$0
TIME (minutes)
Figure
2. Effect of temperature on the increase in adenylate cyclase activity caused by VIP or GMPPNP. Membranes were suspended in 50 mM TrisCl (pH 7.5), 1 mM dithiothreitol, 0.15% (wt/vol) albumin and incubated with no additions (panel A), VIP (panel B), or GMPPNP (panel C) at the indicated temperature for 60 min. At various times during the incubation aliquots were taken to determine adenylate cyclase activity during a 6-min reaction at 37“C. In each experiment each value was determined in duplicate, and results shown are means from four separate experiments. Vertical bars represent 1 SD.
were incubated with lo-’ M VIP at 4”C, adenylate cyclase activity was approximately twice that observed in membranes incubated without VIP and remained constant for 60 min (Figure 2B). In membranes incubated with lo-’ M VIP at 37”C, enzyme activity decreased progressively to less than 10% of the initial value after 45 min (Figure 2B). When membranes were incubated with 10m4 M GMPPNP at 4”C, adenylate cyclase activity was approximately twice that observed in membranes incubated without GMPPNP and remained constant for 60 min (Figure 2C). In contrast to results with VIP, in membranes incubated with 10e4 M GMPPNP at 37”C, enzyme activity increased XI- to 20-fold by 15 min and remained constant thereafter (Figure 2C).
Effect of Removing Activity Measured Incubation
the Agonist on Enzyme During a Subsequent
When membranes were incubated with a particular agent at 4°C and then assayed for enzyme activity at 37”C, the observed activation of adenylate cyclase (Figure 2B and 2C) could have occurred during the incubation at 4’C or during the 6-min assay at 37°C. To examine this possibility membranes were first incubated with 10m7 M VIP for 10 min at 4”C, washed to remove free VIP, and then assayed for adenylate cyclase activity at various times during a second incubation at 4°C or 37’C (Figure 3A). Under these conditions stimulation of adenylate cyclase activity decreased progressively during a 60min second incubation at 37°C and reducing the temperature of the second incubation to 4’C significantly slowed the rate of decrease in VIP-stimulated
enzyme activity (Figure 3A). In membranes which had been first incubated with lo-“ M GMPPNP for 10 min at 4”C, washed, and then incubated at 4’C, GMPPNP-stimulated adenylate cyclase activity was similar in magnitude to that caused by lo-’ M VIP (cf. Figure 3B with Figure 3A) an9 did not change during the second incubation at 4°C (Figure 3B). When the temperature of the second incubation was 37°C instead of 4”C, GMPPNP-stimulated enzyme activity increased five-fold within 15 min and remained constant for the subsequent 30 min (Figure 3B). When membranes were first incubated with GMPPNP at 37°C and then washed, GMPPNP-stimulated adenylate cyclase activity was approximately 13-times greater than it was in membranes first incubated at 4”C, did not change during a second incubation at 4°C and increased slightly when the temperature of the second incubation was 37OC (Figure 3B).
Time-Course for Stimulation of Adenylate Cyclase Activity in Membranes First Incubated with VIP plus GMPPNP To test for a possible interaction bqtween the actions of VIP and GMPPNP on adenylite cyclase activity, membranes were first incubated with both agents for 10 min, washed, and then incubated at 37’C or 4°C (Figure 3C). With VIP plus GMPPNP the pattern of stimulation and of the effect of different incubation temperatures were similar to those seen with GMPPNP alone (cf. Figure 3C with Figure 3B). That is, stimulation of enzyme activity was greater in membranes first incubated at 37OC than it was in membranes first incubated at 4°C and the increase
708
OLINGER AND GARDNER
GASTROENTEROLOGY
in enzyme activity did not change during a second incubation at 4”C, but increased during the first 15 min of a second incubation at 37°C (Figure 3). With membranes first incubated at 4’C, the increase in enzyme activity caused by the two agents in combiprotein/l0 min) was signation (170 & 15 pmol/mg nificantly greater than the additive effects (VIP alone, 19 + 3 plus GMPPNP alone, 20 + 3 = 49 + 7) (mean + 1 SD from three experiments). In addition, in membranes first incubated with VIP plus GMPPNP at 4°C the increase in adenylate cyclase activity during a second incubation at 37’C (130 + 18 pmol/mg protein/l0 min) was significantly greater than that seen with membranes first incubated with GMPPNP alone (80 + 11) (mean f 1 SD from three experiments).
Reversibility Stimulated
of the Decrease in VIPAdenylate CycJase Activity
To examine the reversibility of the decrease in adenylate’cyclase activity in membranes first incubated with VIP, washed, and incubated at 37’C, GMPPNP or VIP was added after adenylate cyclase had reached a minimum value. In membranes first incubated with 10m7 M VIP at 4°C for 10 min, washed to remove free VIP, and then incubated at 37”C, adenylate cyclase activity decreased progressively during the second incubation and by 45 min reached the same value as that in membranes which had been first incubated without VIP (Figure 4). In membranes which had been first incubated with or 28
T
T
[VIP =
A
107
3501
-
Vol. 77, No. 4, Part 1
without VIP, adding the peptide after 45 min of the second incubation caused a three-fold increase in adenylate cyclase activity by 60 min and this increase persisted for the remainder of the second incubation (Figure 4). Adding GMPPNP after 45 min of the second incubation also increased adenylate cyclase activity (Figure 4). However, in contrast to the effect of adding VIP, the increase in adenylate cyclase activity caused by adding GMPPNP to membranes which had been first incubated with VIP was 60% greater than that obtained with membranes which had been first incubated without VIP (Figure 4). Ability of GMPPNP to Modify VIP or Secretin on Adenylate Activity
the Action CycJase
of
To examine further the effects of GMPPNP on the activation of adenylate cyclase caused by VIP or secretin, membranes were incubated with varying concentrations of VIP or secretin with or without lo-“ M GMPPNP, washed, and assayed for adenylate cyclase activity (Figure 5). In membranes incubated with VIP alone a small but significant increase in adenylate cyclase activity could be detected with 3 X lOmy M VIP and maximal stimulation occurred incubated with with 3 x lo-’ M VIP. In membranes VIP plus GMPPNP, as the concentration of VIP increased, VIP-stimulated enzyme activity could be detected at 10-l’ M VIP, increased progressively to a plateau at 3 x lo-” M VIP, and then increased to a
1
Ml
C
600-
24
! GMl+NP = 1W M,; , VIP=lO-7M ,
20
,,‘A 12
.
200 +
-1
150 I
3
i
8,
37O I
109 c41 1\ 0
~2 4O 1
50 i
-_ 30
15
L
370 45
60
0
15
30
45
so
TIME (minutes) Figure 3. Effect of temperature
on the increase in adenylate cyclase activity caused by VIP or GMPPNP. Membranes were suspended in 50 mM T&Cl (pH 7.5), 1 mM dithiothreitol, 0.15% (wt/vol) albumin, and incubated with no additions or the indicated agents for 10 min (first incubation). The membranes were then washed once with 56 vol of iced suspending medium. After centrifugation at 1000 g for 5 min and aspiration of the supernatant. membranes were resuspended to their original volume and incubated at 4’C or 37’C for 60 min (second incubation). At various times during the second incubation aliquots were taken to determine adenylate cyclase activity during a 6-min reaction at 37°C. Values given are for the increase in adenylate cyclase activity caused by the indicated agents and were calculated by subtracting enzyme activity measured at the same time using membranes which were processed identically, but contained no additions. Open symbols, first incubation at WC; closed symbols, first incubation at 4°C. Triangles, second incubation at 37°C; circles, second incubation at 4’C. In each experiment each value was determined
in duplicate, and results shown are means from three separate experimentk
Vertical bars represent 1 SD.
ADENYLATE
32
2 k
Lug
Ei
GMPPNP 1104 MI
4*
\
5 4c
pi
; 0
15
30
- -45
60
75
90
Figure 4. Time-course of adenylate cyclase activity in membranes from pancreatic acinar cells. Membranes were suspended in 50 mM TrisCl (pH 7.5). 1 mM dithiothreitol and 0.15% (wt/vol) albumin and incubated for 10 min at 4’C with or without 10e7 M VIP (first incubation). The membranes were washed once by adding 50 vol of iced suspending medium containing no VIP. After centrifugation at 1000 g for 5 min and aspiration of the supernatant, membranes were resuspended to their original volume with suspending medium and incubated at 37’C (second incubation). Adenylate cyclase activity was determined at different times during the second incubation, using a 6-min reaction at 37°C. After 45 min of the second incubation, VIP or CMPPNP was added, and the incubation was continued for 45 min. Open symbols. first incubation with VIP; closed symbols, first incubation without VIP. In each experiment each value was determined in duplicate, and results shown are means from three separate experiments. Vertical bars represent I SD.
2 %;lsl
240 220 200
240 220 210 200 180 160 140 120 100 80
A ,$,
J
---tl
4
Y
I plus GMPPNP (10-Q Ml
IN PANCREATIC
To examine the ability of VIP to increase in adenylate cyclase activity GMPPNP, membranes were incubated ent concentrations of GMPPNP alone or VIP, washed, and assayed for adenylate
520 484 440 400 360 320 280 240 200
S
plus GMPPNP (104 M)
J
VIP (log M)
Figure
7og
; _.__;_~;--+%“ e -10
-9
-8
-7
SECRETIN (log M)
-6
-5
modify the caused by with differplus lo-’ M cyclase ac-
C
plus VIP (107 M) ‘
*
L-
c
*
i
160
sq 40 ,Y 20 0d n -11
CELLS
Effect of VIP on the Increase in Adenylate Cyclase Activity Caused by GMPPNP
?,
-5
ACINAR
second plateau as the VIP concentration increased from 3 x lo-” M to lo-’ M (Figure 5A). The maximal increase in adenylate cyclase activity caused by VIP was 10 times greater in membranes incubated with VIP plus GMPPNP than it was in membranes incubated with VIP alone (Figure 5A). Results with secretin were similar to those obtained with VIP. In membranes incubated with secretin alone a small, but significant increaSe in adenylate cyclase activity could be detected with lo-” M secretin and enzyme activity increased progressively as the secretin concentration was increased to 3 X lo-” M (Figure 5 B). In membranes incubated with secretin plus GMPPNP, as the concentration of secretin increased, stimulation of enzyme activity could be detected at lo-” M secretin, increased to a plateau at lo-“’ M secretin, remained constant until lo-” M secretin and at secretin-concentration above lo-” M there was a progressive increase in secretin-stimulated adenylate cyclase activity (Figure 5B). As occurred with VIP, the maximal increase in adenylate cyclase activity caused by secretin was 10 times greater in membranes incubated with secretin plus GMPPNP than it was in membranes incubated with secretin alone (Figure 5B).
36
3
CYCLASE
120 80 40 0
i
I
Alone
-6
5
_ :’ -8
-7
4
3
GMPPNP (log M1
5. Effect of VIP, secretin, and GMPPNP on adenylate cyclase activity in membranes from pancreatic acinar cells. Membranes were suspended in 50 mM TrisCl (pH 7.5). 1 mM dithiotheitol and 0.15% (wt/vol) albumin, incubated with the indicated agents for 20 min and then assayed for adenylate cyclase activity during a 6-min reaction at 37°C. Incubations containing GMPPNP were at 37°C whereas incubations without GMPPNP were at 4’C. Values given are for the increase in adenylate cyclase activity caused by the indicated concentration of the particular agent. For example, in panels A and B basal activity (19.6) was subtracted from all values determined with VIP or secretin alone, while activity with GMPPNP alone (297) was subtracted from all values determined with GMPPNP plus VIP or secretin. In each experiment each value was determined in duplicate and results shown are means from three separate experiments. Vertical bars represent 1 SD.
710
OLINGER
Table
1.
AND
GARDNER
GASTROENTEROLOGY
Effect of Secretin5_27 on Adenylase Cyclase Activity in Membranes from Pancreatic Acinar
Effect of Secretin5_z, Activity
Cells Adenylate
cyclase (pmoles cAMP/mg protein/l0 min)
Addition SecretinS-,, (30 PM) VIP (0.1 PM) VIP + Secretin,_,, GMPPNP (0.1 mM) GMj’PNP + VIP GMPPNP + Secretin,.,,
0.1 19.6 2.5 297.0 480.4
+ + f + +
0.2 1.6 0.2 26.7 32.0
271.3 + 18.0
Membranes were suspended in 50 mM TrisCl (pH 7.5), 1 mM dithiothreitol, and 0.15% (wt/vol) albumin, incubated with the indicated agents for 20 min and then assayed for adenylate cyclase activity using a 6-min reaction at 37°C. Assays containing GMPPNP were incubated at 37°C; others were at 4°C. Values given are for the increase in adenylate cyclase activity caused by the indicated agents and were calculated by subtracting enzyme activity measured in incubations which were processed identically, but contained no additions. In each experiment each value was determined in triplicate, and results given are means (fl SD) from four separate experiments.
tivity (Figure 5C). In membranes incubated without VIP, GMPPNP-stimulated enzyme activity could be detected at 3 X lo-’ M GMPPNP and was maximal at 1O-4 M GMPPNP (Figure 5C). In membranes incubated with VIP plus GMPPNP the configuration of the dose-response curve for enzyme activity stimulated by GMPPNP was unaltered, but the increase in enzyme activity caused by a maximally effective concentration of GMPPNP was increased by 60-70% (Figure 5C). Table
2. Effect of Guanyl Cyclase Acinar
Activity Cells
Nucleotides in Membranes
Vol. 77, No. 4, Part 1
on Adenylate
Cyclase
Secretin .5_-27, a C-terminal fragment of secretin which is a competitive antagonist of the action of secretin or VIP on adenylate cyclase activity in pancreatic acinar cells,‘~“~’ did not alter basal adenylate cyclase activity or the stimulation of enzyme activity caused by GMPPNP, but significantly inhibited the increase in cyclase activity caused by VIP (Table 1). Effects Action
of Different Guanyl of GMPPNP
Nucleotides
on
the
The increase in adenylate cyclase activity caused by GMPPNP alone or in the presence of VIP could be inhibited by guanyl nucleotides such as GTP, GDP, and GMP (Table 2) but not by guanosine or guanine (not shown). Not only were these nucleotides able to inhibit the increase in enzyme activity caused by GMPPNP, but they could also reverse the action of GMPPNP on adenylate cyclase activity (Figure 6). In membranes which had been first incubated at 37°C with GMPPNP or GMPPNP plus VIP, washed, and then incubated at 37’C, adding 5 mM GTP or 5 mM GDP during the second incubation caused progressive decrease in enzyme activity (Figure 6).
on Adenylate from Pancreatic
Adenylate cyclase (percent of control)
(10 ILM)
GMPPNP (10 pM) plus VIP (0.1 PM)
100 14 + 2 15 r+ 2 59 + 6
100 16 +2 23 + 3 72 f 4
GMPPNP Addition None GTP (5 mM) GDP (5 mM) GMP (5 mM)
Membranes were suspended in 50 mM TrisCl (pH 7.5). 1 mM dithiothreitol, 0.15% (wt/vol) albumin. To determine adenylate cyclase activity, membranes were incubated for 10 min at 37°C with the indicated agents and washed once by adding 50 vol of iced, suspending medium. After centrifugation at 1000 g for 5 min and aspiration of the supcrnatant, membranes were resuspended to their original volume and assayed for adenylate cyclase activity during a 6-min reaction at 37°C. Values are expressed as percent of that determined with no additions (i.e., with GMPPNP alone or with GMPPNP plus VIP alone). In each experiment each value was determined in duplicate, and results given are means (il SD) from three separate experiments.
Effect of guanyl nucleotides on the activation of adenylate cyclase caused by GMPPNP. Membranes were suspended in 50 mM TrisCl (pH 7.5), 1 mM dithiothreitol and 0.15% (wt/vol) albumin and incubated with GMPPNP or GMPPNP plus VIP for 10 min at 37°C (first incubation). The membranes were then washed by adding 50 vol of iced suspending medium containing no VIP or GMPPNP. After centrifugation at 1000 g for 5 min and aspiration of the supernatant, membranes were resuspended to their original volume with iced suspending medium containing no additions, GDP, or GTP and incubated for 60 min at 37“C (second incubation). At different times during the second incubation samples were taken to determine adenylate cyclase activity using a 6-min reaction at 37°C. In each experiment each value was determined in duplicate, and results shown are means from three separate experiments. Vertical bars represent 1 SD.
0c:tolwr 1979
ADENYLATE CYCLASE IN PANCREATIC ACINAR CELLS
Discussion In agreement with previous studies in other tissUes7.~1 II)ZH:,(I)the present results illustrate that although GMPPNP, secretin, and VIP can activate adenylate cyclase in membranes from pancreatic acinar cells, the characteristics of activation by the guanyl nucleotide differ substantially from those of the activation caused by the peptides. At 37”C, basal as well as VIP-stimulated enzyme activity decreased progressively to a minimum value by 30-45 min whereas GMPPNP-stimulated activity increased and became maximal after 15 min. Reducing the incubation temperature to 4°C abolished the time-dependent decrease in basal and VIP-stimulated activity. From the present studies, we cannot determine whether the time-dependent decrease in basal and VIP-stimulated adenylate cyclase activity observed at 37°C represented loss of some membrane-associatccl cofactor required for enzyme activity or deterioration (i.e., irreversible inactivation) of the membrane preparation. Our finding that after 45 min of incubation enzyme activity could be partially stimulated by adding VIP or GMPPNP, indicates that at least part of the decrease in enzyme activity may have resulted from loss of one or more hypothetical cofactors which are required to support basal as well as stimulated enzyme activity. In agreement with findings in other tissues,“.‘“.‘n-‘” the relatively slow, temperature-dependent activation of adenylate cyclasc in acinar cell membranes incubated with GMPPNP appeared to reflect changes which occurred after nucleotide binding since membranes incubated with GMPPNP and washed to remove free nuclcotidc also showed an increase in adenylate cyclase activity during the first 15 min during a second incubation at 37’C. The ability of GMPPNP to stimulate adenylate cyclase activity and to modify the stimulation caused by VIP or secretin appears to reflect its interaction with the GTP regulatory site since, as has been observed in other systcms,“.‘R.“~‘” the action of the nucleotide analoguc could be inhibited by GTP as well as by GDP and GMP. In some tissues GMPPNP has been found to cause a persistent activation of adenylate cyclase which is not reversible by washing the membranes to remove the free nucleotide or by adding guanyl nucleotides such as GTP or GDP.“.“‘~‘“~‘”In the present studies we found that the action of GMPPNP could not be reversed by washing the membranes, but could be reversed by adding GTP or GDP. These findings illustrate that in membranes from pancreatic acinar cells, guanyl nucleotides which are susceptible to hydrolysis can reverse the persistent activation caused by GMPPNP. Because the molecular basis of action of guanyl nucleotides
711
on adenylate cyclase is not known, it seems unreasonable to speculate about the possible mechanisms by which native guanyl nucleotides are able to reverse the activity of adenylate cyclase caused by GMPPNP. Our findings do indicate that in membranes from pancreatic acinar cells, the association of guanyl nucleotides with the GTP regulatory site do not reflect a simple bimolecular reaction. In agreement with the findings of Svoboda et al.!‘,“’ GMPPNP potentiated the action of secretin and VIP on adenylate cyclase activity in membranes from pancreatic acinar cells, i.e., the increase in adenylate cyclase activity caused by GMPPNP plus VIP or secretin was substantially greater than the sum of the increase caused by each agent acting alone. This potentiation was not attributable to changes in binding of GMPPNP to the GTP regulatory site or of VIP to its receptors since neither agent caused detectable changes in binding of the other agents. In some systems where guanyl nucleotides enhance the activation of adenylate cyclase caused by a hormone or neurotransmitter, the nucleotide has been found to accelerate the rate of dissociation of the bound agonist and by so doing, reduce the amount of agonist bound at the steady state.“,“” Potentiation of the stimulation of adenylate cyclase caused by VIP plus GMPPNP required that the VIP receptor be occupied by an active agonist since potentiation of adenylate cyclase activity did not occur with the guanyl nuclcotide plus secretin, 27 which binds to the hormone of adenyreceptors,‘~“~7 but does not cause activation late cyclase. Finally, potentiation did not require that VIP and GMPPNP be added to the incubation simultaneously. In membranes first incubated with VIP for 10 min at 4”C, washed, and then incubated at 37°C for 45 min, the increase in adenylate cyclase activity caused by adding GMPPNP to the second incubation was substantially greater than that in membranes first incubated without VIP, but otherwise treated identically. In addition to potentiating the action of agents on adenylate cyclase in membranes from pancreas”’ as well as virtually every other tissue studied, guanyl nucleotides can also alter the time-course of activation caused by various agonists (for review, see references 8,15,16,18). When guanyl nucleotides and hormones or neurotransmitters are added to the adenylate cyclase assay medium, several minutes are required to achieve maximal activation by the nucleotide while activation by hormones or neurotransmitters is maximal at the earliest time it is technically possible to take a sample.““‘~‘“~“’ The timecourse of activation of cyclase when a guanyl nucleotide plus a hormone or neurotransmitter is added to the cyclase assay medium is the same as that caused by adding the hormone or neurotransmitter alone. In
712
OLINGER
AND
GARDNER
the present studies the time-course of stimulation of adenylate cyclase by VIP plus GMPPNP was the same as that for the nucleotide alone. This similarity occurred in membranes incubated with both agents as well as in membranes incubated with both agents and then washed before a second incubation and was not altered by changing the incubation temperature. The basis for this difference is not known; however, the present findings as well as results of others illustrate that with or without an agonist, full stimulation of adenylate cyclase by GMPPNP requires at least 15 min of incubation at 37°C. In the present studies, GMPPNP not only potentiated the action of secretin and VIP and modified the time-course of action of the peptides, but also substantially altered the dose-response curves for the action of VIP and secretin. GMPPNP increased the sensitivity to VIP and secretin in that without the nucleotide, peptide concentrations of greater than 1 nM were required to cause detectable stimulation of adenylate cyclase activity, whereas with the nucleotide, significant stimulation of enzyme activity could be detected with peptide concentrations as low as 10 pM. GMPPNP also altered the configuration of the dose-response curves for VIP and secretin; however, VIP did not alter the configuration of the dose-response curve for the action of the guanyl nucleotide. Without GMPPNP, the dose-response curve for the action of VIP or secretin was monophasic whereas with the nucleotide, the doseresponse curves for the peptides were biphasic. In most tissues studied to date guanyl nucleotides have potentiated the increase in adenylate cyclase activity caused by hormones and neurotransmitters, but have not altered the sensitivity to stimulation by an agonist or the configuration of the dose-response curve.“.“-“’ In some systems, however, guanyl nucleotides have been shown to increase the sensitivity of adenylate cyclase to stimulation by various agonists.“~“~‘” Svoboda et al.“’ studying plasma membranes from rat pancreas found that guanyl nucleotidcs potentiated the increase in adenylate cyclase activity caused by peptides, caused a relatively small (approximately 50%) decrease in peptide concentration required to cause half-maximal enzyme activity, but did not alter the configuration of the dose-response curves for the actions of the peptides. The basis for the difference between the results obtained by Svoboda et al.“’ and those in the present studies are probably attributable to the different incubation conditions. The present results indicate that in intact acinar cells the interaction of guanyl nucleotides with the GTP-regulatory site may be an important determinant of the cellular response to VIP and secretin and suggest that GTP is required for optimal expression of the biologic activity initi-
GASTROENTEROLOGY
ated by interaction of agonists with ferring as well as the VIP-preferring
Vol. 77, No. 4, Part 1
the secretin-prereceptors.
References 1. Christophe
JP, Cordon TP, Gardner JD: Interaction of porcine vasoactive intestinal peptide with dispersed pancreatic acinar cells from the guinea pig. Binding of radioiodinated peptide. J Biol Chem 251:4629-4634,1976 2. Robberecht P, Conlon TP, Gardner JD: Interaction of porcine vasoactive intestinal peptide with dispersed acinar cells from the guinea pig: structural requirements for effects of VIP and secretin on cellular cyclic AMP. J Biol Chem 251:4635-4639, 1976 3. Gardner JD, Conlon TP, Fink ML, et al: Interactions of peptides related to secretin with hormone receptors on pancreatic acinar cells. Gastroenterology 71:965-970, 1976 4. Gardner JD, Conlon TP, Beyerman HC, et al: Interaction of synthetic lo-tyrosyl analogs of secretin with hormone receptors on pancreatic acinar cells. Gastroenterology 73:52-56, 1977 JD, Jackson MJ: Regulation of amylase release from 5. Gardner dispersed pancreatic acinar cells. J Physiol270:439-454, 1977 JD, Rottman AJ, Natarajan S, et al: Interaction of se6. Gardner cretm,_,, and its analogues with hormone receptors on pancreatic acini. Biochem Biophys Acta 583:491-503,1979 JD: Effects of cholecystokinin on adeny7. Long, BW, Gardner late cyclase activity in dispersed pancreatic acinar cells. Gastroenterology 73:1008-1014.1977 Y, et al: Role of adenine and 8. Rodbell M, Lin MC, Salomon guaninc nucleotidcs in the activity and response of adenylate cyclase systems to hormones: evidence for multisite transition states. Adv Cyclic Nucl Res 5:3-29,1975 M, Robberecht P, Camus J, et al: Subcellular distri9. Svoboda bution and response to gastrointestinal hormones of adenylate cyclase in rat pancreas. Eur J Biochem 69:185-193.1976 P, Camus J, et al: Association of bind10. Svoboda M, Robbcrecht ing sites for guanine nucleotides with adenylate cyclase activation in rat pancreatic plasma membranes. Eur J Biochem 83:287-297,1978 11. Rodbell M, Birnbaumer L, Pohl SL, et al: The glucagon-sensitive adenyl cyclase system in plasma membranes of rat liver. An obligatory role of guanyl nucleotides in glucagon action. J Biol Chem 246:1877-1882,197l EJM: Activation of pigeon erythrocyte 12. Pfeuffer T, Helmreich membrane adenylate cyclase by guanyl nucleotide analogues and separation of a nucleotide binding protein. J Biol Chem 250:867-876,1975 P, Bennett V, Jacob S: Irreversible stimulation of 13. Cuatrecasas adenylate cyclase activity of fat cell membranes by phosphoramidate and phosphonatc analogs of GTP. J Membr Biol 23:249-278,1975 14. Lefkowitz R: Guanosine triphosphate binding sites in solubilized myocardium-relation to adenylatc cyclase activity. J Biol Chcm 250:1006-loll,1975 M: The catecholamine-responsive adenylate cy15. Schramm clase system and its modification by Sguanylimidodiphosphate. Adv Cyclic Nucl Res 5:105-115. 1975 GD, Speigel AM, Gardner JD: P-adrenergic recep16. Aurbach tors, cyclic AMP, and ion transport in the avian erythrocyte. Adv Cyclic Nucl Res 5:117-132,1975 P, Jacobs S, Bennett V: Activation of adcnylatc cy17. Cuatrecsas clase by phosphoramidate and phosphonate analogs of GTP: possible role of covalent enzyme-substrate intermediates in the mechanism of hormonal activation. Proc Nat1 Acad Sci USA 721739-1743.1975
Octol1r:r
1979
ADENYLATE
18. Londos C, Salomon Y, Lin MC, et al: 5’.guanylimidodiphosphate. a potent activator of adenylate cyclase systems in eukaryotic cells. Proc Nat1 Acad Sci USA 71:3087-3090, 1974 19. Hunter WM, Greenwood FC: Preparation of iodine-131 lahelled human growth hormone of high specific activity. Nature (Lend) 194:495-496.1962 20. Amsterdam A, Jamieson JD: Structural and functional characterization of isolated pancreatic exocrine cells. Proc Nat1 Acad Sci USA 69:3028-30321972 21. Amsterdam A, Jamieson JD: Studies on dispersed pancreatic cxocrine cells. I. Dissociation technique and morphologic characteristics of separated cells. J Cell Biol 63:1037-1056, 1974 22. Amsterdam A, Jamicson JD: Studies on dispersed pancreatic cxocrine cells. II. Functional characteristics of separated cells. J Cell Biol 63:1057-1073, 1974 23. Gardner JD, Conlon TP, Klaevcman HL, et al: Action of cholccystokinin and cholinergic agents on calcium transport in isolated pancreatic acinar cells. J Clin Invest 56:366-375, 1975 24. Gardner JO, Conlon TP, Adams TD: Cyclic AMP in pancre-
25. 26.
27.
28.
29.
30.
CYCLASE
IN PANCREATIC
ACINAR
CELLS
713
atic acinar cells: effects of gastrointestinal hormones. Gastroenterology 70:29-35, 1976 Salomon Y, Londos C, Rodhell M: A highly sensitive adenylate cyclase assay. Anal Biochcm 58:541-548,1974 Klaeveman HL. Conlon TP. Levy AG, et al: Effect of gastrointestinal hormones on adenylate cyclase activity in human jejunal mucosa. Gastroentcrology 68:667-675.1975 Lowry OH, Rosenhrough NJ, Farr AL, et al: Protein measurement with the Folin phenol reagent. J Biol Chem 193265-275, 1951 Rutten WJ, De Pant JJHHM, Bonting SL: Adenylate cyclast: in the rate pancreas. Properties and stimulation by hormones. Biochem Biuphys Acta 274:201-213,1972 Kempen HJM, DC Pont JJHHM. Bonting SL: Rat pancreas adenylate cyclase. 11. Inactivation and protection of its hormone receptor sites. Biochem Biophys Acta 370:573-584, 1974 Milutinovic S, Schulz I, Rosselin G: The interactions of secretin with pancreatic membranes. Biochem Biophys Acta 436:113-127. 1976