(Received for publication, October 11, 1995)
From the
Vasoactive intestinal polypeptide (VIP), pituitary adenylate
cyclase-activating polypeptide-27 (PACAP-27), and PACAP-38 stimulated
insulin release with EC values of 0.15, 0.15, and 0.06
nM respectively, as expected for the VIP2/PACAP3 receptor
subtype. Secretion was stimulated promptly and peaked at 6-10
min. At 30 min, the secretion rate was still 2-3-fold higher than
the control rate. The peptides increased cyclic AMP and
[Ca
]
transiently so
that at 30 min they had returned to control values. Therefore, an
additional signal is required to explain the prolonged stimulation of
release. The prolonged effects, but not the acute effects of VIP and
PACAP on insulin release were inhibited by low concentrations of
wortmannin, a phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor.
While wortmannin inhibited PI 3-kinase activity in cell lysates, no
activation by the peptides was seen. Therefore, the
wortmannin-sensitive pathway is either dependent on basal PI 3-kinase
activity, or another target for wortmannin is responsible for
inhibition of the peptide-stimulated secretion. It is concluded that
the acute stimulation of insulin release by VIP and PACAP is mediated
by increased cyclic AMP and
[Ca
]
, whereas the
sustained release is mediated by a novel wortmannin-sensitive pathway.
Vasoactive intestinal polypeptide (VIP) ()and PACAP
(pituitary adenylate cyclase-activating polypeptide) are members of a
family of peptides that includes secretin, glucagon, and glucagon-like
peptide-1 with diverse actions in several cell
types(1, 2) . Among a large array of biological
effects, they stimulate the secretion of
amylase(3, 4) , the release of hypophysiotropic
hormones such as prolactin(5, 6) , and lower the
systemic blood pressure(7) . Additionally they augment insulin
secretion (8, 9) and are thought to do this by
interaction with the VIP-2/PACAP-3 receptor(10) , which has
been cloned(11) . This receptor binds VIP and PACAP with
similar affinities and has a selective tissue distribution. PACAP
exists in two amidated forms: PACAP-27 and the C-terminally extended
variant PACAP-38(12) . The N-terminal 1-28 sequence of
PACAP-38 shows 68% sequence homology with VIP(13) . PACAP is an
extremely potent modulator of insulin secretion and has been reported
to augment glucose-stimulated insulin release from rat pancreatic
islets at a concentration of 0.1 pM(14) . Both VIP
and PACAP are localized in the gastrointestinal tract, and the pancreas
and VIP- and PACAP-like immunoreactivity was observed in capillaries
and nerve fibres terminating on pancreatic
islets(15, 16) . PACAP-specific staining has been
detected even within the islet in rat(14) .
The VIP/PACAP
family of peptides are thought to couple positively to the adenylyl
cyclase system in their target cells. In some cell types, they increase
[Ca]
. In accordance
with this, in single cell studies in the presence of 8.3 mM glucose, 80% of rat pancreatic
-cells responded to PACAP with
a rise in [Ca
]
, a rise
that was blocked by nitrendipine (14) . However, in the mouse
-cell, VIP-stimulated insulin secretion was not associated with a
detectable rise in cyclic AMP levels(17) , a finding that
suggests the possibility of an action independent of cyclic AMP and
activation of PKA. Furthermore, in AtT20 cells, the stimulation of
-endorphin secretion by VIP was unaffected by the stable
expression of mutated regulatory subunits of the cyclic AMP-dependent
protein kinase, an expression that blocked the effects of isoproterenol
and analogs of cyclic AMP(18) . The authors concluded that VIP
could stimulate
-endorphin secretion by a mechanism that did not
involve cyclic AMP-dependent protein kinase.
In view of these data,
we have reinvestigated the mechanisms of action of these peptides to
stimulate insulin release. The HIT-T15 -cell line was used, and
careful attention was paid to the temporal aspects of the second
messenger responses and their effect on insulin secretion. The results
showed that the peptides increase cyclic AMP and
[Ca
]
levels only
transiently, but they exert a prolonged stimulatory effect on insulin
secretion. The prolonged stimulation of insulin secretion by VIP and
PACAP is manifest after [Ca
]
and cyclic AMP levels have returned to basal values. It is
unlikely therefore that either cyclic AMP or raised
[Ca
]
is responsible
for the prolonged stimulation of release. Finally, it was found that
the prolonged release induced by VIP and PACAP, but not the release
induced by forskolin or glucose, was selectively inhibited by low
concentrations of wortmannin. Wortmannin, a microbial metabolite found
in a variety of fungal species, is known to be an inhibitor of
phosphatidylinositol 3-kinase (PI 3-kinase) with IC
values
between 2 and 4 nM(19) . At higher concentrations,
wortmannin has been linked to the inhibition of phospholipase D (20) and myosin light chain kinase(21, 22) ,
whereas no effect on cAMP-dependent protein kinase was detected at
concentrations of wortmannin up to 10 µM(23) . The
selective inhibition of PACAP- and VIP-stimulated insulin release by
low concentrations of wortmannin points to a novel signal transduction
pathway in the action of these peptides.
For the measurement of insulin release under perifusion
conditions, approximately 3 10
cells were placed in
each of 4 chambers of a perifusion apparatus. Experiments were
performed in KRB buffer at 37 °C at flow rates of 1 ml/min. The
cells were preperifused for 50 min for stabilization of the secretion
rates and then exposed to test and control conditions as appropriate.
Samples were collected at 1-min intervals and kept at -20 °C
until assayed.
Insulin content of the cells was measured after extraction in 1% Triton X-100 and freezing the cells overnight.
Figure 1: Concentration-response characteristics for the effect of VIP and PACAP to stimulate insulin release in the presence of 4 mM glucose. A, the responses to VIP over the range of 0.01 nM to 100 nM. The left two bars illustrate insulin release in the presence of 0.2 and 4 mM glucose alone. The measurements were made under static incubation conditions over 30 min. The results are expressed as means ± S.E. n = 4-13. B, the effects of PACAP-38 over the range of 0.01-100 nM. The left two bars illustrate insulin release in the presence of 0.2 and 4 mM glucose alone. The measurements were made under static incubation conditions over 30 min. The results are expressed as means ± S.E. n = 3-5.
Figure 2: Effects of 10 nM VIP and PACAP on insulin release in the absence of glucose (three left-hand bars) and in the presence of the low glucose concentration of 0.2 mM (three right-hand bars). Results are expressed as means ± S.E. for pg of insulin released/1000 cells/30 min. n = 4-7.
Figure 3:
Temporal profile of the effects of VIP and
PACAP (both 10 nM) to stimulate insulin release. The
experiments were carried out under perifusion conditions in the
presence of 4 mM glucose. Results are expressed as means
± S.E. for pg of insulin released/million cells/min. n = 6. , control;
, PACAP;
,
VIP.
Figure 4: Concentration-response characteristics for the effects of VIP and PACAP-38, over the range of 0.1-100 nM, to increase cyclic AMP levels in the presence of 4 mM glucose. The measurements were made under static incubation conditions after 5 min of exposure to the peptides. The results are expressed as means ± S.E. n = 3-7.
Figure 5:
Effects of 10 nM VIP and 10
nM PACAP in the absence and presence of 3 µM nitrendipine, and of 300 nM forskolin and 300 nM 1,9-dideoxyforskolin on
[Ca]
. The experiments
were carried out in the presence of 4 mM glucose. The
individual traces of fura-2 fluorescence shown are representative of at
least four similar experiments for each peptide and for
forskolin.
Neither
the peptides nor forskolin had any effect on
[Ca]
in the presence of 1 or 3
µM nitrendipine, an L-type Ca
channel
blocker (3 µM shown in Fig. 5). Additionally, the
response was unaffected by depletion of intracellular Ca
stores by thapsigargin (data not shown). These results suggest
that the increase in [Ca
]
is
due entirely to activation of L-type voltage-dependent Ca
channels and increased Ca
entry. Because of
this apparent reliance on the voltage-dependent channels, measurement
of the effects of VIP and PACAP on membrane potential were made using
the potential-sensitive indicator bisoxonol. Only a very slight and
again transient effect to depolarize the membrane was detectable (data
not shown). The concentration-response characteristics of the effects
of VIP and PACAP on [Ca
]
,
measured at the peak of the response, are shown in Fig. 6. VIP
caused a small increase in [Ca
]
at 0.1 nM and a near maximal increase at 1 nM.
The maximum effect was seen at 10 nM. Interestingly, the peak
value for [Ca
]
decreased from
the maximum value as the concentration of VIP was increased further to
100 nM. The results with PACAP-38 were similar to those with
VIP except that PACAP was more potent. PACAP-38 gave a much larger
increase than VIP at 0.1 nM and peaked at 10 nM. As
was the case with VIP, an increase in PACAP-38 from 10 nM to
100 nM caused the [Ca
]
to decrease from the peak value.
Figure 6:
Concentration-response characteristics for
the effect of 0.01 to 100 nM VIP and PACAP-38 to increase
[Ca]
in the presence
of 4 mM glucose. Measurements of
[Ca
]
were made at the
peak of the [Ca
]
responses. Results are expressed in nM as means
± S.E. n =
3-14.
Figure 7: Time courses of the effects of PACAP and 300 nM forskolin to increase cyclic AMP levels in the HIT-T15 cell. A, cyclic AMP levels were measured at 0.5, 1, 5, 10, and 30 min in the absence and presence of 10 nM PACAP. Results are expressed as means ± S.E. n = 4. B, cyclic AMP levels were measured at 0.5, 1, 5, and 30 min in the absence and presence of forskolin. All experiments were in 4 mM glucose. Results are expressed as means ± S.E. n = 4-7.
In view of the importance
of the findings that both [Ca]
and cyclic AMP levels were at basal levels at 30 min, a time at
which insulin secretion was still stimulated by VIP and PACAP, a second
series of experiments was performed to confirm that cyclic AMP levels
had indeed returned to basal after 30 min of exposure to VIP and PACAP.
These data are shown in Table 1. It is clear that at 30 min, the
peptides no longer have any effect on the cyclic AMP content of the
cells.
In contrast to the transient responses to the peptides, forskolin had a prolonged effect on cellular cyclic AMP levels. As can be seen from Fig. 7B, the increase in cyclic AMP caused by forskolin reached a peak at 5 min and only slightly declined thereafter. At 30 min, cyclic AMP levels remained highly elevated.
Given that insulin secretion at 30 min is still strongly stimulated
by both VIP and PACAP and that the peptides no longer have any effects
on [Ca]
and cyclic AMP, it
would appear that VIP and PACAP must activate mechanisms in addition to
cyclic AMP and [Ca
]
in order to
cause a persistent stimulation of insulin release. Consequently,
additional experiments were performed to investigate these mechanisms.
Figure 8:
The effect of 10 nM VIP on
insulin secretion in control and PKC-down-regulated HIT-T15 cells
(treated for 24 h with 300 nM TPA). The experiments were
carried out under perifusion conditions. Results are expressed as means
± S.E. n = 5 for both. A, the release
rates are expressed as pg of insulin/10 cells/min. B, the results for the TPA-treated cells have been normalized
to account for the decrease in insulin content that occurs during the
24-h treatment period.
, control;
, control
(TPA-treated);
, VIP;
, VIP
(TPA-treated).
Under control conditions, in the presence of 4 mM glucose, VIP caused a prompt stimulation of insulin secretion that peaked after 10 min and declined slowly thereafter. The rate of insulin release in the down-regulated cells in 4 mM glucose was less than that of the control cells by approximately 40%. In these cells, stimulation by VIP was essentially similar to that in the controls except that both the starting level and the subsequent peak values were lower.
Treatment
with TPA for 24 h reduces the insulin content of -cells (insulin
content of control cells was 250 ± 10 ng/10
cells,
and that of the down-regulated cells was 147 ± 11 ng/10
cells, i.e. a 41% reduction). Thus it is possible that
the reduced insulin secretion seen was due to the decrease in insulin
content. Indeed, when the rate of insulin secretion is normalized for
the decreased insulin content, as shown in Fig. 8B,
there is no difference in the secretion rates for control or
down-regulated cells. It seems unlikely, therefore, that PKC is
responsible for the prolonged peptide stimulation of insulin secretion.
Figure 9: The effect of wortmannin (WT) to inhibit insulin secretion stimulated by VIP. A, the two left-hand bars show insulin secretion due to 4 mM glucose in the absence and presence of wortmannin. The subsequent bars show the effects of 10 nM VIP alone, and VIP in the presence of 10, 30, and 100 nM wortmannin. The results are expressed as means ± S.E. n = 4. B, the concentration-response characteristics are shown for the effects of wortmannin, over the range of 1-30 nM, to inhibit insulin secretion stimulated by 10 nM VIP. Results are expressed as means ± S.E. n = 4.
Wortmannin had similar effects on insulin secretion stimulated by PACAP-38 as it did on VIP-induced release (see Table 2).
Next, the effect of wortmannin on forskolin-stimulated insulin secretion was studied in paired experiments and compared directly with its effect on the VIP response. The results show that while wortmannin inhibited the VIP response by 37%, as anticipated, it had no effect on the forskolin response (see Fig. 10). Again, there was no effect on the rate of insulin secretion stimulated by 4 mM glucose. Thus wortmannin selectively inhibits VIP- and PACAP-stimulated insulin release at concentrations that have no effect on glucose- or forskolin-stimulated release.
Figure 10: The effect of 100 nM wortmannin (WT) to inhibit insulin secretion stimulated by 10 nM VIP and the lack of effect upon secretion stimulated by 300 nM forskolin or 4 mM glucose. The results are expressed as means ± S.E. n = 4.
Under perifusion conditions, it was
possible to document the effect of wortmannin on the temporal profile
of the VIP and PACAP responses. In experiments (not shown) the effect
of simultaneous application of PACAP and wortmannin was studied. The
effect of PACAP alone was similar to the previous results shown in Fig. 3. There was a prompt stimulation of insulin secretion,
which reached a maximum rate some 3 times the rate in 4 mM glucose alone. After 30 min, the rate was still more than 2-fold
greater than the control. Interestingly, there was no effect of
wortmannin on the initial response to PACAP. Instead, there was a
delayed inhibitory effect that reduced the PACAP-stimulated insulin
release to near basal levels by 20 min. Because of the possibility that
these results were due to a slow onset of action of wortmannin, perhaps
by slow diffusion to its site of action (although this seems unlikely),
the experiments were repeated under conditions such that the test cells
were preincubated with wortmannin for 60 min prior to the stimulation
by peptide. This was done to preclude any concerns about possible
delayed access of wortmannin to its site of action. The results
obtained under these conditions with VIP and wortmannin are presented
in Fig. 11. Under control conditions, 10 nM VIP
stimulated insulin secretion as anticipated with a rapid increase in
secretion rate to a peak value and then a small decline to a plateau
after 25 min. At the 30-min point, the secretion rate was still 3 times
that of the untreated (4 mM glucose) controls. In the presence
of wortmannin, and despite long exposure to the drug, the response to
VIP was still unaffected for the first 10 min. At that time, the rate
of VIP-stimulated insulin release stopped increasing, inhibition of
release began, and the release rate was approaching basal values after
a further 10 min. Thus the inhibitory effect of wortmannin was exerted
on the persistent secretion stimulated by VIP and not on the acute
stimulation. This is in accord with an action of VIP (and PACAP) to
stimulate insulin secretion acutely by means of the prompt but
transient increases in cyclic AMP and
[Ca]
and to cause persistent
secretion by virtue of a wortmannin-sensitive pathway.
Figure 11:
The effect of 100 nM wortmannin
on insulin secretion (4 mM glucose) and 10 nM VIP-stimulated secretion. Wortmannin was added 50 min prior to
zero time on the figure. The experiments were carried out under
perifusion conditions, and the results were expressed as pg of
insulin/10 cells/min (mean ± S.E.). n = 5.
, control;
, VIP;
, WT control;
, WT + VIP.
The next
studies performed in the course of this work were aimed at the
measurement of PI 3-kinase activity. The results of typical experiments
are shown in Fig. 12. In Fig. 12A, PI 3-kinase
activity is shown after incubation of the cells with 4 mM glucose alone (lane 1) and with PACAP-38 and VIP (lanes 3 and 5). No stimulation of PI 3-kinase
activity by PACAP or VIP was detected in this and three similar
experiments. Wortmannin (100 nM) markedly inhibited PI
3-kinase activity in the presence of glucose, PACAP, and VIP (lanes
2, 4, and 6). In Fig. 12B, PI
3-kinase activity is shown in the presence of both 0.2 and 4 mM glucose (lanes 1 and 4). There was no difference
in PI 3-kinase activity in the two glucose concentrations (n = 3). PACAP-38 and VIP, both of which stimulate insulin
secretion in the presence of either of these glucose concentrations,
did not change the activity of PI 3-kinase (lanes 2, 3, 5, and 6). Finally, in panel C,
the effects of different concentrations of wortmannin (3-100
nM) on PI 3-kinase activity in the presence of 4 mM glucose can be seen. Wortmannin caused a concentration-dependent
inhibition of the enzyme (n = 3). Near total inhibition
was achieved by 100 nM wortmannin (n = 9). In
another approach for testing the effects of PACAP and VIP on PI
3-kinase activity, HIT cells were labeled with PO
and then treated with PACAP, VIP, or, as a control, 4 mM glucose alone. Phosphoinositides were extracted from the cells,
separated by thin-layer chromatography, and autoradiographed. No
differences in the
P-labeling patterns, indicative of
increased PI 3-kinase activity, were detected, n = 6.
Figure 12:
Autoradiographs of thin-layer
chromatography plates of PI 3-kinase assays. Immunoprecipitated PI
3-kinase was assayed with PI and P-[ATP] as
described under ``Experimental Procedures.'' Cells were
preincubated for 30 min prior to the test conditions. When wortmannin
was used, it was present during the preincubation as well as the test
period. All additions listed here for the individual lanes refer to the
15-min treatment of the cells prior to lysis and immunoprecipitation of
the PI 3-kinase. The numbers refer to the lanes and identify the
experimental conditions imposed. Panel A, lane 1, 4
mM glucose (4G); lane 2, 4G + 100 nM wortmannin (WT); lane 3, 4G + 10 nM PACAP-38; lane 4, 4G + PACAP-38 + WT; lane
5, 4G + 10 nM VIP; lane 6, 4G + VIP
+ WT. Panel B, lane 1, 0.2 mM glucose
(0.2G); lane 2, 0.2G + 100 nM PACAP-38; lane
3, 0.2G + 100 nM VIP; lane 4, 4G; lane
5, 4G + 100 nM PACAP; lane 6, 4G + 100
nM VIP. Panel C, lanes 1 and 2, 4G; lanes 3-6: 4G + wortmannin at 3, 10, 30, and 100
nM.
The results of these studies show that the effects of VIP and
PACAP to stimulate insulin secretion are the result of a complex set of
interconnected second messenger systems. Both peptides interact with
receptors that are positively linked to adenylyl cyclase to raise the
cellular cyclic AMP content. This, in turn, increases the activity of
the -cell L-type voltage-dependent Ca
channels
and increases [Ca
]
. The
increased [Ca
]
and cyclic AMP
then synergize to stimulate insulin secretion. It seems likely from our
data that both VIP and PACAP interact with the same receptor on the
-cell, because the simultaneous addition of both peptides did not
increase the rate of insulin release to levels higher than the addition
of either one alone (data not shown). Furthermore, from the relative
potencies of the peptides, it can be assumed that the HIT-T15 cell
expresses the VIP type 2/PACAP type 3 receptor. This assumption is in
accord with the observation that mRNA for this receptor subtype is
expressed at moderate levels in the HIT-T15 (hamster) cells, RINm5f
(rat) cells and in rat pancreatic islets and at high levels in the MIN6
(mouse) cell (11) .
The effects of the peptides were rapid,
and increases in cyclic AMP were detected after only a 30-s exposure,
the earliest time point measured. Increased cyclic AMP levels in RINm5F
cells, and increased adenylyl cyclase activity in RINm5F and RIN14B in
response to VIP have been reported
previously(29, 30) . The increase in
[Ca]
peaked also at 30 s. In
the presence of a stimulatory concentration of glucose (4 mM),
the peptides induced a prompt increase in insulin secretion. It is
obvious that any agent that increases both cyclic AMP and
[Ca
]
under these conditions
will stimulate insulin secretion. However, it was noted in these
studies that the effects of VIP and PACAP to stimulate insulin
secretion were large and prolonged, whereas the effects of the peptides
on cyclic AMP content and on [Ca
]
were transient. Additionally, the effects on
[Ca
]
were quite small. After a
30-min exposure to the peptides, there was a complete dissociation
between the stimulated rate of insulin release and the levels of cyclic
AMP and [Ca
]
. At this time,
while insulin release was still strongly stimulated by the peptides,
both the cyclic AMP content and [Ca
]
had returned to basal values. These data point to the existence
of at least one additional signal transduction mechanism by which
insulin secretion is maintained at stimulated levels. In seeking this
additional mechanism, we were unable to find evidence that would
implicate the activation of PLC or PKC as mechanisms by which the
peptides might increase secretion rates. Neither down-regulation of PKC
nor inhibition of PKC (data not shown) blocked the response to the
peptides. Additionally, the increase in
[Ca
]
in response to VIP and
PACAP was abolished by nitrendipine, and this would not have occurred
if there was activation of PLC and mobilization of intracellular
Ca
. While this is in agreement with data obtained in
rat islets, where the increase in
[Ca
]
in response to PACAP was
also blocked by nitrendipine(14) , this result contrasts with a
previous report in the HIT-T15 cell, where it was suggested that PACAP
was acting also through mobilization of intracellular
Ca
(31) . Indirect evidence for a coupling of
PACAP to the PLC pathway was obtained in Xenopus oocytes
transfected with PACAP type 3 receptors. These cells responded to PACAP
with activation of calcium-activated Cl
currents(11) , a phenomenon that was also observed with
glucagon-like peptide-1 in transfected COS-7 cells(32) . These
results may be due to excessively high levels of receptor expression.
The major finding in the present study is that the sustained
stimulation of insulin release by VIP and PACAP is extremely sensitive
to wortmannin, a known inhibitor of PI 3-kinase. The best two
characterized isoforms of PI 3-kinase are heterodimers consisting of an
85-kDa regulatory and a 110-kDa catalytic subunit(33) , which
are stimulated by a variety of growth factor receptors. This includes
translocation and tyrosine phosphorylation of the 85-kDa subunit.
Subsequently, the 110-kDa subunit gains access to its substrate and
phosphorylates phosphatidylinositol at the D-3 position of the inositol
ring. However, PI 3-kinase can also be activated by heterotrimeric G
protein linked receptors without the need for tyrosine phosphorylation
of the 85-kDa subunit (34) and there are reports that PI
3-kinase can be directly regulated by G protein and
subunits(35, 36) . The lipid products of this enzyme
reaction, PtdIns[3,4,5]P
and
PtdIns[3,4]P
have been implicated in mitogenesis,
cell transformation, and importantly in
exocytosis(37, 38) . It was because of the latter that
we investigated the effects of wortmannin on insulin secretion. Our
measurements of PI 3-kinase activity show that wortmannin inhibits the
enzyme over the same range as it inhibits PACAP- and VIP-stimulated
insulin secretion. However, no stimulation by the peptides or by
glucose was detected. The lack of effect of glucose was not unexpected
as wortmannin had no effect on glucose-stimulated release. Therefore,
from the data presented, there are at least three possibilities with
respect to the nature of the wortmannin-sensitive pathway, which is
linked to the stimulation of exocytosis. The first is that basal levels
of PI 3-kinase activity are required for the provision of downstream
intermediates necessary at some point of convergence with the VIP/PACAP
signaling pathway. The second is that there may be a specific
``exocytosis-associated'' PI 3-kinase activity, which is
undetectable by the techniques used here because of the preponderance
of the other PI 3-kinase activities. The third possibility, despite PI
3-kinase being the only enzyme thus far reported to be inhibited by
wortmannin at these low concentrations, is that the wortmannin effect
is exerted on another enzyme which is critical in the
stimulus-secretion pathway for these two peptides and not on PI
3-kinase. However, if a PI 3-kinase is involved in this pathway, it is
noteworthy that in retinal pigment epithelial cells, VIP stimulated the
phosphorylation of pp60
, an oncogene protein
kinase that is known to phosphorylate PI 3-kinase(39) . Thus a
potential signal transduction pathway exists between the VIP receptor
and pp60
, which leads to PI 3-kinase.
Subsequent steps to increased exocytosis are unknown and need to be
investigated.
From the patterns of release seen during peptide
stimulation in control and wortmannin-treated cells, the temporal
effects of the different components of the stimulatory mechanisms can
be observed. It appears that the initial response, the prompt upswing
of stimulated insulin release is due to the combined effects of
increased [Ca]
, which can
trigger release, cyclic AMP, which enhances the stimulated level of
release, and the wortmannin-sensitive signal. It is interesting that
cyclic AMP, well known to be a potentiator of release rather than an
initiator(40, 41) appears to be responsible for the
increased channel activity that results in increased
[Ca
]
and enhances release by at
least two mechanisms under these conditions. After the stimulation of
release by VIP in the presence of wortmannin, the increased rate of
release is not sustained and decays in a manner consistent with the
short time course of the effects of VIP and PACAP on cyclic AMP and
[Ca
]
. It supports also the idea
that the wortmannin-sensitive pathway is primarily involved in the
prolongation of the stimulation of insulin release by these two
peptides. Neither the nature of this wortmannin-sensitive pathway nor
the mechanism by which the occupied VIP/PACAP receptors activate the
pathway is known. The activation is not secondary to the elevation of
cyclic AMP or to the increase in [Ca
]
because the effect of forskolin on insulin release, which mimics
the actions of the peptides on cyclic AMP and
[Ca
]
, is not inhibited by
wortmannin. However, the wortmannin-sensitive pathway is
receptor-mediated, because the prolonged stimulation of release decays
rapidly to base-line values after removal of the peptides. The link
between the receptor and this novel wortmannin-sensitive mechanism by
which stimulated insulin secretion is maintained remains to be
determined. It could be mediated by
or
subunits of
G
, which is activated by VIP and PACAP. It could
conceivably be mediated by a heterotrimeric G protein not currently
known to be associated with the VIP-2/PACAP-3 receptor. While the low
concentrations of wortmannin used to inhibit the responses of the
peptides are in accord with an action on PI 3-kinase, there may well be
other kinases in the cell that have a similar sensitivity to wortmannin
and which are, perhaps, essential components of this
-cell
stimulus-secretion pathway.