It has been reported that the synthetic heptapeptide cholecystokinin (CCK) analogue JMV-180 evokes cytosolic Ca2+ signals in pancreatic acinar cells via mechanisms that do not include either the generation or action of inositol 1,4,5-trisphosphate (InsP3) (Saluja, A. K., Dawra, R. K., Lerch, M. M., and Steer, M. L. (1992) J. Biol. Chem. 267, 11202-11207; Yule, D. I., and Williams, J. A. (1992) J. Biol. Chem. 267, 13830-13835). We have now investigated the CCK- and JMV-180-evoked cytosolic Ca2+ oscillations by measurement of the Ca(2+)-sensitive ion currents in internally perfused mouse pancreatic acinar cells. We find that the InsP3 receptor antagonist heparin (500 micrograms/ml) blocks Ca2+ oscillations induced by both CCK (5-20 pM) and JMV 180 (10-40 nM), whereas de-N-sulfated heparin (500 micrograms/ml), which does not affect InsP3 binding to its receptor, fails to inhibit the responses to the two agonists. We conclude that the cytosolic Ca2+ oscillations evoked by both CCK and JMV-180 are dependent on functional InsP3 receptors

Petersen, O. H.

Thorn, P.

University of Queensland eSpace

THE JOUKSAI UF BIOLWK A L
0 1993 by The American 
. CHEMISTRY 
Society for Blochemistry and Molecular Blology. Inc 
Vol. 268, No. 31, Issue of November 5, pp. 23219-23221.  1993 
Printed In U.S.A. 
Calcium Oscillations in Pancreatic Acinar  Cells, Evoked by the 
Cholecystokinin  Analogue JMV-180, Depend  on  Functional  Inositol 
1,4,5-Trisphosphate  Receptors* 
(Received for publication, April 1, 1993, and  in revised  form,  July 16, 1993) 
Peter Thorn and Ole H. Petersen 
From the Medical Research Council Secretory Control Research Group, the Physiological Laboratory, University of 
Liuerpool, l? 0. Box 147, Liuerpool L69 3BX, United Kingdom 
It has been  reported  that the synthetic  heptapeptide 
cholecystokinin (CCK) analogue JMV-180 evokes cytoso- 
lic Ca2+ signals in pancreatic acinar cells via mecha- 
nisms  that  do  not  include either the generation  or  action 
of inositol 1,4,54risphosphate  (InsP,)  (Saluja, A. K., 
Dawra, R. K., Lerch, M. M., and  Steer, M. L. (1992) J.BioZ. 
Chem. 267, 11202-11207; Yule, D. I., and Williams, J. A. 
(1992) J.  Biol. Chem. 267,13830-13835). We have now in- 
vestigated the CCK- and  JMV-180-evoked cytosolic  Ca2+ 
oscillations by measurement of the Ca2+-sensitive ion 
currents in internally  perfused  mouse  pancreatic  acinar 
cells. We find that the InsP,  receptor  antagonist  heparin 
(500 pg/ml)  blocks  Ca2+ oscillations induced by both CCK 
(5-20 PM) and JMV 180 (1040 n M ) ,  whereas  de-N-sulfated 
heparin  (500 pg/ml), which  does  not  affect  InsP,  binding 
to  its receptor, fails to inhibit  the  responses to the  two 
agonists. We conclude that the cytosolic Ca2+ oscilla- 
tions evoked by both CCK and  JMV-180 are  dependent 
on  functional  InsP3  receptors. 
Cytosolic  Ca2+  oscillations  evoked by hormones and neuro- 
transmitters  are  generally  mediated  by  the  intracellular mes- 
senger inositol 1.4,5-trisphosphate (InsP3)l (1). In pancreatic 
acinar cells, the two main agonists acetylcholine (AChi and 
cholecystokinin  (CCK)  evoke cytosolic Ca2+ signals that can  be 
inhibited  by  intracellular  application of the InsP,  receptor an- 
tagonist heparin (2, 3) or by the phospholipase C inhibitor 
U73122 (4). It has  been  reported that the  synthetic  heptapep- 
tide  CCK  analogue JMV-180 releases  intracellular Ca2+ from 
an InsP,-independent  store i n  permeabilized  pancreatic  acinar 
cells,  since  this  analogue could evoke Ca2+ release  in the pres- 
ence of a high heparin concentration ( 5 ) .  In agreement  with 
this, Yule and Williams (4) found that the phospholipase C 
inhibitor  U73122  cannot  inhibit cytosolic Ca2+ oscillations 
evoked by JMV-180. These  findings  suggest that JMV-180 
evokes a Ca2+ signal via mechanisms  that do not  include  either 
the generation or action of InsP, (4, 5). It is,  however,  difficult 
to understand the apparent difference in the mechanism of 
action of CCK and JMV-180,  since, at the low agonist  concen- 
trations required to evoke cytosolic Ca2+ oscillations,  both 
agents  are  supposed  to  interact  with the same class of high 
affinity  CCK  receptors (6). 
* This work  was supported by a Medical Research Council  program 
grant. The costs of publication of this article were defrayed in part by 
marked “aduertisernent” in accordance with 18 U.S.C. Section 1734 
the payment of page charges. This article must therefore be hereby 
solely t o  indicate this fact. 
ACh, acetylcholine; CCK, cholecystokinin; JMV-180, synthetic hepta- 
The abbreviations used are: InsP,, inositol 1,4,5-trisphosphate; 
peptide CCK analogue JMV-180; [Ca2+Ii, cytosolic free calcium  ion  con- 
centration. 
We have now investigated the CCK- and JMV-180-evoked 
cytosolic CaZ+ signals by measurement of the  Ca2+-sensitive  ion 
currents  in  internally  perfused  mouse  pancreatic  acinar cells. 
We find that the InsP, receptor  antagonist  heparin blocks both 
CCK- and JMV-180-induced Ca2+ oscillations, whereas de-N- 
sulfated heparin, which does not affect InsP, binding to its 
receptor (7,8) fails  to  inhibit  the  responses  to he two  agonists. 
We conclude that the cytosolic Ca2+ oscillations  evoked by both 
CCK and JMV-180 are  dependent  on  functional InsP3 recep- 
tors. 
MATERIALS AND METHODS 
Cell Preparation-Isolated single mouse pancreatic acinar cells and 
small cell clusters were prepared by pure collagenase (Worthington) 
digestion in the presence of trypsin inhibitor (4 mg/ml;  Sigma  type 1.1-S) 
as described  previously (9). 
Solutions-The standard extracellular solution contained (mM): 140 
NaC1,4.7  KC1,1.13 MgCI,, 11 glucose, 1 mM Ca2+, 10 HEPES-NaOH, pH 
7.2. The standard intracellular solution (pipette solution in whole  cell 
experiments) contained (miv): 140 KC1, 1.13 MgC12, 0.1-0.2 EGTA, 2 
ATP, 10 HEPES-KOH, pH 7.2. Bath solution changes at the cell took 
less than 5 s (10). Two heparin preparations (one of molecular  weight < 
3000 and one with a mean M ,  of 12,000), de-N-sulfated heparin, and 
cholecystokinin  were obtained from Sigma. JMV-180 was kindly given 
to us by Dr. J. Martinez (Montpellier). 
Experimental Protocol-Experiments were all carried out at room 
temperature (20-25 “C). Standard whole  cell recording techniques were 
used (10, 11). Patch pipettes of between 2  and  4 megohms  were  pulled 
from microhematocrit (Assistent) tubes. Seals of >10 gigohms were 
produced on the cell membrane, and gentle suction or voltage pulses 
often  formed  whole  cell  recordings assessed by an increase in capaci- 
tance and noise. Series resistance was measured by the compensation 
circuitry of the LIST  EPC7 amplifier, and whole  cell  recordings  were 
rejected if the series resistance was greater than 15 megohms. The 
whole  cell current records  were obtained by voltage steps from a holding 
potential of -30 mV to a potential of 0 mV. Each potential was  held  for 
150 ms, and the stepping frequency  was 3 Hz. At the resolution shown 
in  the figures, the two current traces obtained at the two holding po- 
tentials appear continuous. Using our solutions, the reversal potential 
of both the CI- and nonselective cation currents  are at 0 mV. Small 
deviations in Ecl and E,,,,,, and in the holding potential sometimes 
produce small inward or outward currents at 0 mV. At -30 mV  we 
obtain a measure of both the Ca2+-dependent currents that correlates 
well  with the [Ca2*I, signal (9, 12). 
The pulse  protocol and data storage were carried out using a program 
developed by Smith (13) with a 286 IBM-compatible  microcomputer and 
a Cambridge  Electronic Devices analogue to digital converter. 
In  the figures  shown (except Fig. 11, the current traces are  taken from 
the start of the whole  cell configuration. Therefore, the left-hand edge of 
the traces indicates the start of the equilibration of the pipette contents 
with the cell  cytoplasm. In  the case of large molecules  such as heparin, 
equilibration would  be expected  to take a relatively long time (14). In 
our experiments, effective concentrations of heparin are reached within 
about 5 min. 
Ca2+-dependent Currents-The experiments described here use the 
Ca2+-dependent ionic currents to monitor changes in [Ca2+],. These 
currents have been  shown  to accurately reflect  local and global [Ca2+], 
signals (12). Heparin has no direct effect  on these currents as shown in 
23219 
23220 Calcium  Oscillations in 
experiments where currents directly activated by intracellular Ca2+ 
infusion could not be blocked by intracellular  heparin  application (2). 
RESULTS 
The Effects of CCK-Fig. L4 shows the typical current re- 
sponse to 15 PM CCK previously described (3, 10, 15). Such 
responses were obtained using 5-20 PM CCK in  the  present 
series of experiments. Both the  short spikes and  the broader 
transients  are  due  to  increases  in [Ca2+Ii; however, whereas  the 
short  spikes  are associated  with  a rise  in  [Caz+Ji exclusively in 
the secretory pole, [Ca2+li is elevated throughout  the cell during 
the broad transients (12). When heparin (M, = 3000) was  pre- 
sent in the  pipette solution a t  a  concentration of 500 pg/ml, the 
response to CCK was abolished ( n  = 3). We also tried a heparin 
preparation with  a higher molecular weight (mean molecular 
weight about 12,000) and,  using a  concentration of 500 pg/ml, 
found that  within  10 min after  establishment of the whole cell 
recording  configuration the CCK response  was  abolished in 3 
out of 4  experiments. 
The Effects of CCK-JMV-l8@-Fig. lB shows the effect of 10 
nM JMV-180. Effects corresponding to those  seen  with 5-20 PM 
CCK were obtained using 1 0 4 0  nM JMV-180 (n = 14). The 
quoted  concentration ranges of the  agonists reflect cell to cell 
variations in sensitivity. We used concentrations in each ex- 
periment required to  obtain oscillatory responses. The relative 
potencies of  CCK and JMV-180 found in our experiments  are  in 
agreement  with  those found earlier (5, 16). 
Heparin Effects on JMV-180-evoked Responses-We used two 
protocols to determine  the effect of heparin on Ca2+ mobiliza- 
tion by JMV-180. In one type of experiment, heparin was in- 
cluded in the patch pipette solution at the concentrations 
shown. After forming  a whole cell, the  contents of the  patch 
pipette are in continuity with the cell interior. In previous 
experiments  (2),  it  has been shown in our cells that effects of 
heparin applied from the  pipette solution can be observed after 
about 3-4 min. The  main  limit  to diffusion is  the access resist- 
ance of the  patch, which was  always  monitored, and we rejected 
cells where this  resistance increased  (see “Materials  and Meth- 
ods”). To monitor the  entry of heparin  into  the cell, brief appli- 
cations of ACh were  applied, as  in  the example of Fig. 2 A ,  and 
the reduction  in the response  was observed. It  is  clear  that,  at 
the point  where the calcium-dependent currents in  response to 
ACh are abolished, a subsequent application of JMV-180 fails 
A: CCK 
15 pM CCK 
response to cholecystokinin (15 PM, A)  and JMV-180 (10 n M ,  B ) .  
FIG. 1. Whole cell patch-clamp current oscillations obtained in 
The upper  line of each record was  obtained at 0 mV holding  potential, 
the  reversal  potential of the C1- and  cation  currents.  The lower  line was 
obtained at a holding  potential of -30 mV and  represents  the  combina- 
tion of the calcium-dependent C1- and nonselective cation currents. 
Small  deviations  in Ecl and ECati,, give rise  to  small  currents a t  0 mV. 
Pancreatic Acinar Cells 
500 pg/ml heparin 
B: mouse JMV-180  JMV-180  JMV-180
rat JMV-180 JMV-180  JMV-180 - - __ 
FIG. 2. Current records obtained in the presence of 500 pg/ml 
heparin in the patch pipette. A, repeated  applications of ACh were 
used to  monitor the  heparin-induced  antagonism of the  InsPB receptor. 
Subsequent  application of JMV-180 failed  to  induce  current  activation. 
E ,  repeated  application of JMV-180 demonstrates block  of Ca2+-depend- 
ent  currents. C,  the  same  type of experiment as  in B but  carried  out 
using  acutely  isolated rat  rather  than mouse pancreatic  acinar cells. 
to elicit a  response (n = 3).  In  the second protocol (Fig. 2B), we 
simply observed the effect of repeated applications of JMV-180 
(20 nM) during  the period immediately after  establishment of 
the whole cell configuration. The  experiments  demonstrate a 
rapid abolition of the response (n = 3). 
The previous studies concluding that  the effect of JMV-180 is 
not mediated by InsP, (4, 5) were carried  out on pancreatic 
acinar cells from rats  rather  than mice; therefore, we also per- 
formed a few experiments using rat cells. Fig. 2C shows an 
example of an  experiment  in which heparin blocked the JMV- 
180  (40 nM) induced current oscillation (n  = 3). 
Effects of De-N-sulfated Heparin-Although the evidence 
showing that  heparin is an  InsP, receptor antagonist  is very 
extensive and convincing (17), this  agent may also inhibit  InsP3 
formation (1). A G-protein-uncoupling action of heparin  has 
been described for muscarinic receptors in cardiac myocytes 
(18) and a-adrenoceptors in hepatocytes (19). To control for 
possible nonspecific actions of heparin, we used de-N-sulfated 
heparin, which has been shown not to bind to the InsP3 recep- 
tor (8). Using a concentration of 500 pg/ml de-N-sulfated  hep- 
arin,  the effects of  5-20 PM CCK were not  reduced,  even more 
than  10 min after establishment of the whole cell recording 
configuration (n = 3). Similar  results were obtained  with 10-40 
nM JMV-180, and Fig. 3 shows an example of a  recording  where 
JMV-180 still evoked a response more than 15 min after de-N- 
sulfated  heparin gained access to  the cell interior (n = 3). 
DISCUSSION 
Our experiments show that  the cytosolic CaZ+ oscillations 
evoked by  CCK and  the  synthetic CCK analogue JMV-180 can 
be blocked by intracellular application of the  InsP3 receptor 
antagonist  heparin,  whereas de-N-sulfated heparin, which has 
been shown not  to bind to InsPs receptors (7,8), failed to inhibit 
responses to both these agonists. The most straightforward 
conclusion to be drawn from these  experiments  is  that both the 
CCK- and JMV-180-evoked cytosolic Ca2+ oscillations are de- 
pendent on functional InsP3 receptors. It does not  necessarily 
follow from this that the actions of CCK and JMV-180 are 
initiated by an  increase  in  the  intracellular InsP,  concentra- 
tion. 
The sulfhydryl reagent thimerosal  induces cytosolic Ca2+ OS- 
cillations in both pancreatic  acinar cells (20)  and HeLa cells 
(21)  without elevating InsP3 levels  (21), but  the effects of thi- 
merosal are abolished by heparin (20,  22). This  has led to  the 
Calcium  Oscillations  in  Pancreatic Acinar  Cells 23221 
500 @ml de-N-sulphated heparin 
JMV-180 JMV-180 
FIG. 3. Whole cell current  record  obtained in response to 40 n~ 
JMV-180. In this experiment the patch pipette contained 500 pg/ml 
de-N-sulfated  heparin,  which does not  act  as a  competitive  antagonist 
a t  the InsP,  receptor  and  fails to block the  currents. 
proposal that InsP,  receptor sensitivity  to InsP, can be regu- 
lated  and  that thimerosal and oxidized glutathione  act by in- 
creasing  the  sensitivity of the InsP,  receptor to such  an  extent 
that  the  resting  InsP, level becomes effective in  releasing Ca2+ 
(1). 
There  is much evidence indicating  that agonist-evoked cyto- 
solic Ca2+ oscillations are mediated by a Ca2+-induced Ca2+ 
release mechanism (2, 9, 17, 23), which could in  principle de- 
pend on either InsP, or ryanodine  receptors (1). With regard to 
the actions of CCK and JMV-180, our  data  indicate a role for 
the  InsPs receptors.  Since it  has not been possible to demon- 
strate InsP, formation in response to CCK concentrations 
lower than 100 PM or to concentrations of JMV-180 below 1 VM 
(161, it  is possible that  the cytosolic Ca2+  oscillations are  initi- 
ated by a Ca2+ release process not  dependent on InsP, genera- 
tion. An action  mediated entirely by sensitization of InsP,  re- 
ceptors to  the  resting InsP, level, similar to that described for 
thimerosal,  is one possibility. Another  InsP,  receptor-independ- 
ent Ca2+ release  pathway could also be involved, since exper- 
iments on permeabilized pancreatic  acinar cells show that high 
concentrations of JMV-180 (10-100 PM), but not CCK, can 
evoke Ca2+ release from an InsP3-insensitive  Ca2+ store (5). 
Further work on the mechanism by which CCK receptor 
interaction initiates cytosolic Ca2+ signals is clearly needed, 
but our results show that functional InsPs receptors are in- 
volved in mediating  the cytosolic Ca2+ oscillations. 
REFERENCES 
2. Wakui, M., Osipchuk, Y. V. and  Petersen, 0. H. (1990) Cell 63,  1025-1032 
1. Berridge, M. J. (1993) Nature 361,315-325 
3. Wakui, M., Kase, H.. and  Petersen, 0. H. (1991) J .  Membr: Eiol. 124, 179-187 
4. Yule, D. I.,  and Williams, J. A. (1992) J.  Eiol. Chem. 267,  13830-13835 
5. Saluja,A. K., Dawra, R. K., Lerch, M. M., and  Steer, M. L. (1992) J.  Eiol.  Chem. 
6. Matozaki, T., Martinez, J., and Williams, J .  A. (1989) Am. J .  Physiol. 257, 
7. Ghosh, T. K., Eis, P. S., Mullaney, J. M., Ebert, C. L., and Gill, D. L. (1988) J. 
8. Tones, M. A., Bootman, M. D., Higgins, B. F., Lane, D. A,, Pay, G. F., and 
267,  11202-11207 
G594"600 
Bioi. Chem. 263,  11075-11079 
9. Osipchuk, Y. V., Wakui, M., Yule, D. I., Gallacher, D. V., and  Petersen, 0. H. 
Lindahl, V. (1989) FEES  Lett. 252, 105-108 
(1990) EMEO J .  9.  687-704 
10. Thorn, P., and  Petersen: 0. H. (1992) J. Gen. Physiol. 100,  11-25 
11. Hamill, 0. P., Marty,  A,,  Neher,  E.,  Sakmann, B., and  Sigworth, F. J. (1981) 
12. Thorn, P., Lawrie, A. M., Smith, P., Gallacher, D. V., and  Petersen, 0. H. (1993) 
13. Smith, P. M. (1992) J. Physiol. 446, 72P 
14. Pusch, M., and  Neher, E. (1988) Pfliigers  Arch. Eur. J. Physiol. 411,  204-211 
15. Petersen, C .  C. H., Toescu, E. C., and Petersen, 0. H. (1991) EMEO J. 10, 
16. Matozaki, T., Goke, B., Tsunoda, Y., Rodriguez, M., Martinez, J., and Williams, 
17. Ferris, C. D., and  Snyder, S. H. (1992)Annu. Reu. Physiol. 54,469-488 
18. Ito, H., Takikawa, R., Iguchi, M., Sugirnoto, T., and Kurachi,Y. (1990) Pfliigers 
19. Dasso, L. L. T., and Taylor, C. W. (1991) Eiochem. J .  280,  791-795 
20. Thorn, P., Brady, P., Llopis, J., Gallacher, D. V., and Petersen, 0. H. (1992) 
21. Bootrnan, M. D., Taylor, C. W., and  Bemdge, M. J. (1992) J.  Eiol. Chem. 267, 
22. Missiaen, L., Taylor, C. W., and  Berridge, M. J. (1991) Nature 352,241-244 
23. Petersen, 0. H. (1992) J.  Physiol. 448,  1-51 
~~. ~,~ 
Pfliigers Arch. Eur. J .  Physiol. 391,  85-100 
Cell 74,  661-668 
527-533 
J. A. (1989) J. Eiol.  Chem. 265,6247-6254 
Arch. Err: J. Physiol. 417,  126-128 
Pfliigers  Arch. Eur: J. Physiol. 422,  173-178 
25113-25119 


Calcium oscillations in pancreatic acinar cells, evoked by the cholecystokinin analogue JMV-180, depend on functional inositol 1,4,5- trisphosphate receptors

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Calcium oscillations in pancreatic acinar cells, evoked by the cholecystokinin analogue JMV-180, depend on functional inositol 1,4,5- trisphosphate receptors

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