BY JAIPAUL SINGH but it markedly potentiated the ACh-evoked amylase secretion and ...

14 Pages·2005·1.48 MB·English

J. Phy8iol. (1985), 358,pp. 469-482 469
With7text-figurem
PrintedinGreatBritain
MECHANISM OF ACTION OF INSULIN ON ACETYLCHOLINE-EVOKED
AMYLASE SECRETION IN THE MOUSE PANCREAS
BY JAIPAUL SINGH
From the M.R.C. Secretory Control Group, Department ofPhysiology,
University ofLiverpool, P.O. Box 147, Liverpool, L69 3BX
(Received 9 July 1984)
SUMMARY
1. The effects of insulin and acetylcholine (ACh) on amylase secretion, trans-
membrane movement of45Ca2+ and K+, membrane potential and cyclic nucleotide
levels in the isolated mouse pancreas were investigated.
2. Insulinalonehadnoeffectoneitheramylasesecretionor45Ca2+fractionalefflux
but it markedly potentiated the ACh-evoked amylase secretion and significantly
reduced the ACh-induced 45Ca2+ fractional efflux. These effects were dose related.
3. Insulin evoked a small membrane hyperpolarization and an increase in K+
efflux. The islethormone had virtually noeffectonACh-induced membrane depolar-
ization but it markedly enhanced the ACh-elicited K+ efflux.
4. Both insulin and ACh had marked time-dependent effects on the metabolism
ofadenosine 3', 5'-cyclic monophosphate (cyclic AMP). Insulin increased and ACh
decreased cyclicAMPconcentration whenappliedseparately. However, whenadded
together,insulinandAChcausedarapidandsustainedelevationofcyclicAMPlevels.
5. Superfusion of mouse pancreatic fragments with an exogenous lipid-soluble
derivative ofcyclic AMP (dibutyryl adenosine 3', 5'-cyclic monophosphate) caused
dose-dependent increases in amylase secretion. Dibutyryl cyclic AMP also markedly
enhanced, in a dose-dependent manner, the ACh-evoked amylase secretion.
6. It is concluded that insulin-may exert its potentiating action on ACh-evoked
amylase output in the mouse pancreatic acinar cells by elevating both cytoplasmic
Ca2+ and cyclic AMP levels.
INTRODUCTION
In the exocrine pancreas there are two functionally distinct pathways by which
secretagogues canstimulateenzymesecretion (Gardner, 1979; Schulz & Stolze, 1980;
Petersen, 1982; Williams, 1984). The actions ofcholera toxin, vasoactive intestinal
polypeptide (VIP), secretin and catecholamines areassociated with changes incyclic
AMPlevelswhereasacetylcholine(ACh)cholecystokinin(CCK)-likeandbombesin-like
peptides are associated with changes in membrane potential, release ofcellular Ca2+
and an increase in intracellular cyclic GMP concentration. Two secretagogues (e.g.
ACh and VIP) acting via the two functionally distinctpathways can potentiate one
another whereas two secretagogues (e.g. ACh and CCK) acting through the same
mechanism cannot. The biochemical basis for thispotentiation is still unresolved. It
470 J. SINGH
is believed that the interactions occur at a step distal to the secretagogue-induced
changesincellularcyclicAMPmetabolismandCa2+ mobilization (Jensen& Gardner,
1981) involving protein phosphorylation (Burnham & Williams, 1982; Roberts &
Butcher, 1983).
Recently, several studies have demonstrated that insulin alone does not directly
stimulate enzyme secretion (Danielsson, 1974; Couture, Dunnigan & Morisset, 1972;
Kanno & Saito, 1976) but it can markedlypotentiate the secretaryresponsestoACh
andCCKinthe pancreas (Kanno & Saito, 1976; Saito, Williams& Kanno, 1980a, b).
The cellular mechanism underlying the insulin enhancement ofsecretagogue-evoked
enzymesecretioninthepancreasisstillunclear. Specificinsulinreceptorsarepresent
in pancreatic acinar cells (Korc, Sankaran, Wong, Williams & Goldfine, 1978;
Sankaran,Iwatmoto, Korc,Williams&Goldfine, 1981)andithasbeensuggestedthat
insulin may act to either increase cellularCa2+ mobilization (Saito etal. 1980a, b) or
stimulate the activity ofthe membrane Na+-K+-ATPase transport pump (Kanno,
1975).
Thepresentstudywasdesignedtoinvestigatefurtherthenatureofthepotentiating
actionofinsulinonACh-evokedamylasesecretion. Theapproachwastomeasurethe
effectsofinsulinaloneandin combinationwithAChonenzymesecretion, membrane
potential, transmembrane movements of 45Ca2+ and K+ and endogenous cyclic
nucleotide levels. A preliminary account ofsome aspects ofthis work was presented
to the Physiological Society (Singh, 1983).
METHODS
All experiments were performed on isolated segments ofmouse pancreas. Adult animals were
killed by a blow to the head and the pancreas was quickly removed and placed in a modified
Krebs-Henseleit solution of the following composition (mM): NaCl, 103; KCI, 4*7; CaCl2, 2-56;
MgC12, 1-13; NaHCO3, 25; NaH2PO4, 1-15; D-glucose, 2-8; Napyruvate, 4-9; Nafumarate, 2-7 and
Na glutamate, 49. The solution was gassed with 95% 02-5% C02 and maintained at 37 0C.
Amytase output measurement
The mousepancreaswas cutinto small segments (5-10mg) and atotalweightofabout 150mg
wasplacedinaPerspex flowchamberandsuperfusedwithKrebs-Henseleitsolutionataflowrate
of 1 ml min-'. The amylase concentration in the effluent from the chamber was measured using
an on-line fluorometric assay by the methods ofRinderknecht & Marbach (1970) and Matthews,
Petersen & Williams (1974). Throughout an experiment the generation offluorescence, which is
a linear function ofamylase concentration, was continuously monitored on a pen recorder. The
secretagogue-evokedamylaserelease(i.e.thatabovebasaloutput)wasroutinelyexpressedinterms
oftheoutputatthepeakoftheresponseinu. ml-'(100mgtissue)-'.Oneunitofamylaseisdefined
astheamountofamylasewhichwillliberate 1-0mgofmaltosefromstarchin3minatpH6-9and
at 200C. In this study a-amylase (Sigma type IIA) was used as a standard for calibration. ACh,
insulin and dibutyryl cyclic AMP were added directly to the superfusing solution in known
concentration.
Membranepotentialmeasurement
A single segment ofmouse pancreas was secured to a translucent Perspex platform in a tissue
bath (volume = 10ml) and superfused with oxygenated Krebs-Henseleit solution at a flow rate
of20 ml min-' at 37 0C. Conventional intracellular recordings ofacinar membrane potential and
resistance were made using the methods of Iwatsuki & Petersen (1977, 1978). A single glass
micro-electrode filled with 3 M-KCl and 10mM-K citrate, with tip resistanceofaround20-30MCI,
was inserted into an acinar cell to record membrane potential. The potential was displayed on a
EFFECTS OF INSULIN AND ACh ON MOUSE PANCREAS 471
Tektronix dual-beam oscilloscope and a pen chart recorder. The tissue was stimulated with ACh
and insulin by adding them to the fluid flowing through the bath.
45Ca2+ efflux measurement
Pancreatic segments (100-150 mg) were incubatedfor60 minin a25 ml conical flask containing
20,sCi 45Ca2+ in 2 ml Krebs-Henseleit solution. The flask was shaken at 60 times min' and
maintainedat37 'C. Afterthisloadingperiodthetissuewastransferredtoa 1 ml Perspex chamber
and superfusedwithKrebs-Henseleitsolutionataflowrateof1 ml min- andmaintainedat37 'C.
Following a 120-150 min equilibration period (Matthews, Petersen & Williams, 1973), effluent
sampleswere collected at2 minintervalsdirectlyintoscintillation vials. Thetissuewasstimulated
with ACh and insulin byaddingthem to the fluid flowingthrough the chamber. Attheendofeach
experiment a 10 ml aliquot of scintillant (Triton X-100: toluene 1:1 and containing Scintol 2,
50 ml 1-1; Koch-light Lab Ltd.) was added to each ofthe samples which were then measured for
45Ca2+ content by liquid scintillation analysis (Packard Tricarb 300). The final 41Ca2+remainingin
the tissue at the end of each experiment was measured after the pancreatic segments had been
dissolved in 2 ml Soluene-350 (Packard) and the radioactivity counted in 10 ml scintillation
mixture.
The readings ofradioactivity were then used to calculatevaluesfor 45Ca2+ fractionalefflux using
the following equation: Ax/Atxt where Ax represents the d.p.m. in the time interval At and xt the
tissue4lCa2+ contentatthemid-pointoftheinterval At. Fractionaleffluxisexpressedasafunction
oftime and thus has units ofmin-.
K+ efflux measurement
The pancreas was cut into small segments (3-5 mg) and a total weight of50-80 mg was placed
into a Perspex flow chamber (volume = 0-5 ml) and superfused with Krebs-Henseleit solution at
aflow rate of 110pul min- at 37 'C. The effluentfrom the flow chamber passed to an on-line flame
photometer (Corning-480) for measurements ofthe concentration ofK+ at 1 min intervals. Values
fornetchangeinK+concentrationswereexpressedin mmol 1-1 (100 mgtissue)-' InsulinandACh
were added directly to the superfusion fluid.
Cyclic nucleotide measurement
Approximately 100-150 mgofmousepancreaticsegmentswereplacedineachofseveral Perspex
flow chambers (volume = 1 ml) and superfused with Krebs-Henseleit solution at a flow rate of
1 ml min' at 37 'C. In initial time-course experiments up to ten chambers were used; with five
of these used as test chambers and the remaining five tissue chambers served as control
preparations. Thus, each test preparation had acontrol 'partner' preparation with which it could
be compared. At various precise times following the onset ofeither ACh or insulin superfusion in
known concentration, both test and partner preparations (i.e. chambers and tissues) were frozen
rapidly in liquidnitrogen. In dose-relatedexperimentsdesigned toyield mean valuesforaspecific
time after the onset of stimulation, only four tissue chambers were used. In these cases one
preparation servedasacontrol forthreetestpreparationssinceallfourwerefrozensimultaneously
at the pre-determined time.
Precise details of the cyclic nucleotide extraction and assay procedures have been given
previously (Flitney & Singh, 1980). Frozen tissues were pulverized in a stainless-steel pestle and
mortar (also cooled in liquid nitrogen) and subsequently extracted in acidic ethanol (1 ml 1 N-HCl
in 100 ml absolute ethanol). The samples were evaporated to dryness in a stream ofdry nitrogen,
and the solid residue obtained was dissolved in Tris-EDTA buffer (05 M-Tris, pH 75, containing
4 mM-EDTA). Cyclic nucleotide levels in the resulting solution were assayed using Radiochemical
Centre (Amersham, England)assaykits. DetailsforcyclicAMP, basedonaspecificprotein-binding
method,andforcyclicGMP,aradioimmunoassayprocedure, aregivenintheRadiochemicalCentre
publications TRK 432 and TRK 500 respectively. Total protein was estimated using the Biuret
method (Gornall, Bardawill & David, 1949) and cyclic nucleotide concentrations aregiven inpmol
(mg protein)-'. All values are expressed as the difference in cyclic nucleotide between test and
control preparations.
472 J. SINGH
RESULTS
Effects ofACh and insulin on amylase secretion
In the presence of 10-6 M- and 10-8 M-insulin, amylase output from superfused
segments ofmouse pancreas was 2-57+0-25 u. ml-' (100 mg tissue)-', (n = 33) and
2-59+030 u.ml-' (100 mgtissue)-', (n = 45),respectively.Thecontrolbasalamylase
secretionbeforeinsulinapplicationwas2-61+0-29 u.ml-' (100 mgtissue)-', (n = 78).
2-0u.ml-'
A (100mgtissue)'[
5 mi
a b '--- C
° IAnsCuAhin 10-7M AhI-1n07sM uliACnh 1100--76MM
10-6M
11
10 B 7
9 7
E~~8~0 ~9 ~~10~8~ 10-7 a0-6 ns10
7
4-, 9
C.
E~~~~~~~~~~~~
4
10-9 10-8 10-7 10-6 10-5
Concentration ofACh (M)
Fig. A.A,effectsof10-6 M-insulinincombination with 10-7 M-ACh (a), AChalone (b) and
AChir. siecontinuingpresenceofinsulin(c;sameasa)onamylasereleasefromsuperfused
mouse pancreatic fragments. The tissue was pre-treated with insulin for 5min before
stimulation with ACh. The responses (a-c) are continuous recordings from a single
experiment.Thehorizontalbarsindicatethedurationofstimulation.Verticalcalibration:
2-0u. ml-' (100mg tissue)-'. The horizontal line labelled 0 represents the fluorescence
readingwithout pancreatic tissuein the flow chamber. B, dose-response curves showing
theeffectsofACh(10-9-10-5 M)onamylasesecretionintheabsence(0-0)andpresence
of 10-8 M-(A-A) and 10-6M-(@-*) insulin. Each point is mean+S.E. ofmean, n is
indicated by the numbers besideeach mean point.
The data show that insulin alone had no significant effect (P > 0-95) on amylase
secretion. However, insulin markedly enhanced the ACh-evoked amylase secretion.
Fig. 1A shows original chart recordings of responses produced by superfusing a
preparationwith 1o-7 M-AChinthecontinuingpresenceof10-6 M-insulin (a), without
insulin (b) and subsequently again in the presence of insulin (c). Exposure of
pancreatic segmentsto 10-7 M-AChresulted inamean (+S.E. ofmean) peakamylase
EFFECTS OFINSULINAND ACh ONMOUSEPANCREAS 473
secretion above basal levels of 3-40+0-31 u. ml-' (100 mg tissue)-' (n = 9). In the
presenceofinsulintheACh-inducedamylasesecretionincreasedto6-63+0-41 u.ml-'
(100 mg tissue)-' (n = 9). These effects of ACh were dose related. Fig. lB shows
dose-response curves for the ACh-evoked amylase secretion in the absence (0) and
presence of 10-6 M-(@) and 10-8 M-(A) insulin. The dose-response curves obtained
in the presence ofinsulin were displaced to the left indicating the marked enhance-
ment by insulin ofACh-elicited amylase secretion.
Insulin 10M
0 A
> p ACAh1073M 7s ACh1O7M
E
_40L
Insulin 10-6M
ACh5X1O-7M 30s ACh5X10-7M
E
-45
Fig. 2. Effects ofsuperfusion with ACh and insulin on acinar membrane potential and
resistance. A, effects of 10-7 M-ACh in the presence and absence of 10-6 M-insulin B,
effects of 5x 10-7 M-ACh in the absence and presence of 10-6M-insulin. The responses
A and B are micro-electrode recordings from two different cells. Rectangular hyper-
polarizing current pulses (1 nA, 100msduration) were repetitively injected through the
micro-electrode recording the membrane potential. ACh and insulin were added to the
superfusing solution fortheperiods indicated bythe horizontal bars.
What, then, is the explanation for this apparent potentiating effect? It is well
known that the ACh-evoked enzyme secretion is associated with changes in cellular
Ca2+ metabolism and acinar cell depolarization. It was therefore decided to test the
ability ofinsulin alone and in combination with ACh to alter acinar cell membrane
potential and Ca2+ and K+ mobilization.
Effect ofACh and insulin on membranepotential and Ca2+ and K+ efflux
Fig. 2 shows changes in membrane potential evoked by insulin and ACh. The
resting membrane potential of mouse pancreatic acinar cells in this series of
experiments was -32 3+05 mV (n = 21). Exposure of the preparation to 10-6 M-
insulin for 2 min resulted in a gradual but significant increase in the membrane
potential to a value of -34*4+05 mV (P < 01005; n = 21). In the continuing
presence ofinsulin, 10-7 M-ACh evoked a depolarization of5-30+0025 mV (n = 14)
while exposure to 10-7 M-ACh alone (Fig. 2A) resulted in a depolarization of
4-98+020 mV (n = 14). Fig. 2B shows the effect of 5x 10-7 M-ACh on acinar
membranepotentialintheabsenceandpresenceofinsulin.ThisconcentrationofACh
applied alone induced a depolarization of714+0-22 mV (n = 7). In the presence of
insulin the ACh-elicited depolarization was 7-64+0-33 mV (n = 7). There was no
significant difference (P < 035) between the ACh-induced membrane depolarization
in the presence or absence ofinsulin.
Fig. 3 shows the effects ofinsulin and ACh on the fractional efflux of4-Ca2+ from
pre-loaded pancreatic fragments. ACh (10-5 M) alone caused a transient increase in
45Ca2+ fractional efflux (Fig. 3A: 0). Pre-treatment ofthe preparation with insulin
474 J. SINGH
6 minbeforeAChstimulationhadnoeffecton4SCa2+efflux. However, whenAChwas
introduced in the continuing presence ofinsulin (either 10-6 M: A or 10-8 M: 0) the
ACh-evoked 45Ca2+ efflux was significantly (P < 0-001) reduced (Fig. 3A). Dose-
response curves for the ACh-induced 45Ca2+ fractional efflux in the absence (@) and
presence (0) of10-6 M-insulin are shown in Fig. 3B. These results show that insulin
markedly attentuates the ACh-evoked 45Ca2+ outflow.
003 A
-
ACh1O-M
E_
s
0*02
0
x
U-
0 10 20 30 40 50 60
Time(min)
0015 88 AChalone
Ej0
+
co
> 0.010
wo
°°<-ilACh+10-insulin
i E °
co
ULL
10-9 10-8 10-7 10-6 10-S
ConcentrationofACh (M)
Fig. 3. A, effectsof 10-5 M-ACh in the absence (@-@) and presence of 10-8M-(O-O)
and 10-6M-(A-A) insulin onthefractional efflux of4"Ca21 from pre-loaded superfused
fragments ofmouse pancreas. Each point is the fractional efflux for a 2min collection
periodplottedasafunctionoftime.Insulinwasadded6minbeforestimulationwithACh
and it remained in the perfusing solution throughout ACh application. ACh was added
tothesuperfusateasindicatedbythehorizontalbarfor 12 min. Eachpointismean+S.E.
ofmean, n = 7. B, dose-response curves showing the effectsofACh (10-9-10-6 M) in the
absence (@) and presence (0) of 10-1 M-insulin on the fractional efflux of46Ca22. Each
point is mean +s.E. ofmean, n = 7.
Fig. 4 shows the effect ofACh (10-7 M: * and 10-5 M: 0) on the mean (+S.E. of
mean) net change ofK+ concentration in the effluent in the absence (A and C) and
presence (B) of10-6 M-insulin. Stimulation ofmouse pancreatic fragments with ACh
resulted in a marked and reversible increase in K+ efflux. Addition of 10-6 M-insulin
also caused a small increase in K+ outflow. However, in the continuing presence of
EFFECTS OFINSULIN AND ACh ON MOUSEPANCREAS 475
insulinAChelicitedasignificantincrease (P < 0-005)inK+effluxascomparedtoACh
alone.
Effects ofinsulin and ACh on cyclic nucleotide levels
Mean basal concentrations oftissue cyclic AMP and cyclic GMP were 3-85+0-45
and 0-42+0-03 pmol (mg protein)-' (n = 46), respectively. There was some small
fluctuation in control levels of cyclic nucleotides during the different experimental
conditions, however, their levels remain virtually constant during a particular time
Insulin 10-6M
0-4 ACh ACh ACh
0.-
'Lo 0-2 -
z~~~ C
0-1
-0-1
A
0 10 20 30 40 50 60 70 80 90
Time(min)
Fig. 4. Effects of 1o-6 M-(O) and 10- M-(@) ACh on net changes in K+ concentrations
intheabsence(AandC)andpresence (B)of10-6 M-insulinintheeffluentofasuperfusion
chamber containing segments of mouse pancreas. ACh and insulin were added to the
superfusingmediumasindicatedbythehorizontalbars.Eachpointismean+s.E.ofmean,
n= 7.
courseordose-dependentexperiment.Thecyclicnucleotideconcentrationwasalways
expressedasthedifferencebetweentheconcentrationinthetestandthecorresponding
control preparation. Each data point in Figs. 5 and 6A therefore represents the
difference between two single measurements.
Fig. 5 shows time-course changes in tissue cyclic AMP levels (A) and cyclic GMP
(B) during responses to 10-6 M-insulin (-) and 10-7 M-ACh (E). Insulin evoked a
rapidincreaseincyclicAMP, risingto amaximumofaround 2-5 pmol (mgprotein)-'
after8-12 s.Thisinitialrisewasthenfollowed byanabrupttransientfall, toaround
-2-6 pmol (mg protein)-1 after 25-35 s. Cyclic AMP levels then rose again to reach
a secondary maximum ofabout 7-4 pmol (mg protein)-' after 3-4 min and persisted
at that level for a further 7-8 min. The early rapid rise and fall in cyclic AMP levels
wasaccompaniedbyatransientincreaseincyclicGMPconcentrationswhichreached
a peak of2-5 pmol (mg protein)-' above basal after 15-25 s. The cyclic GMP levels
then declined gradually, to just below the basal level after 60-90 s and stayed there
throughout the remainder of the response. The time-dependent changes in cyclic
nucleotideconcentrationsevokedbyAChwere both quantitatively and qualitatively
different from those seen in response to insulin. The cholinergic agonist induced a
476 J. SINGH
relativelyslowdeclineincyclicAMPlevels, reachingavirtualplateauofaround -35
to -4-0 pmol (mg protein)-' after 2-3 min and remained there throughout the
response. The levels of cyclic GMP rose gradually to reach a maximum of around
1P2-1P5 pmol (mg protein)-' after 2-3 min and persisted above the control value for
up to 6-7 min. In this series of time-course experiments a total of fifty-eight
preparations from twenty-nine mice were used.
8- A
7
-
6-
-C 5-
> 4
0 -
cm 3 -
E _
0.y
CL
-6 20- 1 f=
Fig. 5. Time-course changes in tissue concentrations ofcyclic AMP (A) and cyclic GMP
(B) during superfusion ofmouse pancreatic fragments with 10-6 M-insulin (fl-U) and
10-7 M-ACh (El-E)- Concentrationsareexpressedinpmol (mgprotein)-' andeachpoint
represents the test minus control values at a precise time following onset ofstimulation.
Mean+SE. of mean control levels of cyclic AMP and cyclic GMP were 4-04+0-22 and
044±0802 pmol (mg protein)'l respectively, n = 29.
The latter results clearly demonstrated that both insulin and ACh have marked
opposing influences on cyclic AMP metabolism. The question which now arises is:
what is the nature ofthe time-dependent changes in levels ofcyclic AMP when both
insulin and ACh are combined? In an attempt to investigate this question, the
preparation was first pre-treated with 10-6 M-insulin for 5 mmn (time when insulin-
evoked cyclic AMP level was maximal) before the addition of ACh. Insulin also
remained in the superfusing medium throughout the remainder ofthe response. The
time course of changes in cyclic AMP (0) and cyclic GMP levels (@)are shown in
Fig. 6A. In the continuing presence of insulin, ACh (10-7 M) evoked a rapid and
sustained elevation in cyclic AMP concentrations, rising to around 25-3-0 pmol (mg
protein)-' within 15-20 s. The rapid rise in cyclic AMP levels was accompanied by
EFFECTS OFINSULINAND ACh ONMOUSE PANCREAS 477
A 106 M-insulin
0. CyclicAMP
3
_ 3
00
C D. "
E~E E
.o E 0
O-.
C
10
E~
5 - B I1-5
1-2 'Ca
2 o6
=
0 oXm
c
E0 3 0.9 E 0
E c E .'
1 CL E
0-6
~so' 2;
2
CL
03 c,
ACh+ 101 M-insulin
0
Li
I I I l
10-9 10-o 10-7 10-6 10-
ConcentrationofACh (M)
Fig. 6. A, effectsofinsulin (10-6 M) incombinationwithACh (10-7 M) ontime-dependent
changes in levels ofcyclic AMP (0) and cyclic GMP (0) in mouse pancreatic segments.
Eachpointrepresentsthe cyclicnucleotide concentrations (testminus control values) at
aprecise pointfollowing the onset ofstimulation. The preparation was pre-treated with
insulin for 5min before addition ofACh. B, dose-response curves showing the effects of
ACh (10-9-10-6 M) in the continuing presence of 10-6 M-insulin on cyclic AMP (0) and
cyclicGMP(0)levels. Allvaluesexpressedastestminuscontrollevelsandpreparations
weresuperfusedfor2-3minbeforefreezing.Eachpointismean+s.E.ofmean,n= 3.The
levels of cyclic AMP and cyclic GMP in control preparations were 3*66+0-24 and
0A40+0-02pmol (mgprotein)-', respectively (n 17).
=
aslowtransient increaseincyclicGMPconcentrations, reachingaround 1-{)1-2 pmol
(mg protein)-' after 2-3 min. Thereafter, cyclic GMP declined slowly but remained
above the basal level after 5 min. Fig. 6B shows the dose dependency
(10-9-10-5 M-ACh) ofthe changes in levels in cyclic AMP (0) and cyclic GMP (@)
in the continuing presence of 10-6 M-insulin. All measurements were made 2-3 min
afterapplicationofACh.Inthisseriesofexperiments totalofforty-fourpreparations
a
from twenty-two mice were tested.
478 J. SINGH
Effects ofdibutyryl cyclic AMPand ACh on amylase secretion
In this series ofexperiments the effects ofexogenous dibutyryl cyclic AMP onthe
ACh-evoked amylase secretion was investigated. Superfusion of mouse pancreatic
fragments with dibutyryl cyclic AMP resulted in a clear increase in amylase output.
The mean (±s.E. of mean) peak increases in amylase secretion above basal levels
evoked by 10-4 M-, 5X 10-4M- and 10-3M-dibutyryl cyclic AMP were 0-22+0-02
A
2-0u.mlV 5mi
a (00mgtissue)'[-,
ACh107mb
0- A- 1-7 A~PM
5X10-4M-DBcyclicAMP ACh 1M 5X10-4M-DBcyclicAMP
11
o10 8 7
99 8
8-
g 7
7~~~~
6~~~~~~~
=i=5~~~
QX4 -
gi 3 7
> 2 5
01O5
10- 1O 1 10
10 9 lo-,, 10-7 106 lo-,
Concentration ofACh (M)
Fig. 7. A, effects of 10-7 M-ACh in the presence (a and c) and absence (b) of5x 10-4 M-
dibutyryl (DB) cyclic AMP on amylase release from mouse pancreatic fragments. The
responses(a-c)arecontinuousfluorescenceintensityrecordingsfromasingleexperiment.
The horizontal barsindicate theduration ofstimulation. Vertical calibration 2-0u. ml-'
(100mg tissue)-'. The horizontal line labelled 0 represents the fluorescence reading
withoutpancreatic tissuein the flow cell. B, dose-response curvesshowingtheeffects of
differingconcentrationsofACh(10-9-10- M)onpeakamylasesecretionabovebasallevels
in the absence (0-0) andpresence (@-@) of5x 10-4 M-dibutyryl cyclic AMP. Each
pointis mean+s.E. ofmean, nis shown besideseach point.
(n = 5), 0-52+0-03 (n = 32) and 1-28+0-12 (n = 5) u. ml-' (100 mg tissue)-', respec-
tively. Fig. 7A showsoriginal chartrecordingsofamylase outputfroman individual
experiment. The preparation was superfused with 10-7 M-ACh in the continuing
presenceof5x 10-4 M-dibutyrylcyclicAMP (a),withAChalone (b)andsubsequently
withAChinthepresenceofdibutyrylcyclicAMP (c).Themean (±S.E. ofmean)peak
increase in amylase output above basal levels evoked by 10-7 M-ACh was 3-17+030
(n = 7) u. ml-l (100 mgtissue)-'. Inthepresence of5x 10-4 M-dibutyryl cyclic AMP
the ACh-induced amylase secretion was6-78+030 (n = 7) u. ml-' (100 mg tissue)-.