(Received for publication, July 17, 1995)
From the
Various forms of cross-talk between the Ca and
cAMP signal transduction systems can occur in animal cells depending
upon the types of adenylyl cyclases present. Here, we report that
Ca
oscillations can be generated by hormone
stimulation of type III adenylyl cyclase expressed in HEK-293 cells.
These Ca
oscillations are apparently due to the
unique regulatory features of type III adenylyl cyclase, which is
stimulated by hormones and inhibited by elevated Ca
in vivo. Ca
oscillations were generated by
glucagon, isoproterenol, or forskolin stimulation of type III adenylyl
cyclase and were dependent upon the activity of cAMP- and
calmodulin-dependent protein kinases. Ca
oscillations
were not solely dependent upon cAMP increases since dibutyryl cAMP or (S
)-cAMP did not stimulate Ca
oscillations. We hypothesize that stimulation of type III
adenylyl cyclase leads to increased cAMP, activation of inositol
1,4,5-trisphosphate receptors, and elevation of intracellular
Ca
. As free Ca
increases, type III
adenylyl cyclase activity is attenuated by CaM kinase(s) and
intracellular cAMP levels decrease. When cAMP levels drop below a
threshold level, the inositol 1,4,5-trisphosphate receptor is
dephosphorylated and Ca
is resequestered. This cycle
is repeated if type III adenylyl cyclase is chronically exposed to an
activator. This unique mechanism for generation of Ca
oscillations in cells is distinct from others documented in the
literature.
In most mammalian tissues the Ca and cAMP
signal transduction systems are tightly coupled, and cross-talk between
these two regulatory systems may play an important role for various
physiological phenomena including synaptic plasticity (Xia et
al., 1991; Choi et al., 1993b). Intracellular free
Ca
(Ca
) (
)can affect cAMP levels by modulation of adenylyl cyclase
or phosphodiesterase activities (reviewed by Choi et al. (1993a) and Beavo and Reifsnyder(1990)). On the other hand,
cAMP-dependent protein kinase (PKA) or cAMP can affect
Ca
by regulating Ca
ion channel activity (reviewed by Hell et al.(1994)).
Because of the regulatory diversity of adenylyl cyclases,
phosphodiesterases, and protein kinases, different patterns of
cross-talk between the Ca
and cAMP regulatory systems
may be established in specific cell types.
cDNA clones for eight
adenylyl cyclases have been isolated, and each of these enzymes has
distinct regulatory properties (Krupinski et al., 1989, 1992;
Bakalyar and Reed, 1990; Feinstein et al., 1991; Gao and
Gilman, 1991; Ishikawa et al., 1992; Katsushika et
al., 1992; Yoshimura and Cooper, 1992; Cali et al., 1994;
Watson et al., 1994). Five of these enzymes: I-AC (Tang et
al., 1991; Choi et al., 1992b), III-AC (Choi et
al., 1992a; Wayman et al., 1994), V-AC and VI-AC
(Yoshimura and Cooper, 1992; Katsushika et al., 1992), and
VIII-AC (Cali et al., 1994), are regulated by
Ca. I-AC and VIII-AC are stimulated by intracellular
Ca
in vivo (Choi, 1992b; Cali et
al., 1994), whereas V-AC and VI-AC are inhibited by
Ca
.
III-AC is stimulated by Ca and calmodulin (CaM) in isolated membranes when the enzyme is
also activated by G
(Choi et al., 1992). However,
we recently discovered that Ca
inhibits
hormone-stimulated III-AC activity in vivo (Wayman et
al., 1995). Ca
inhibition of III-AC is not due
to activation of G
or protein kinase C and is apparently
mediated by one of the CaM kinases. For example, Ca
inhibition of III-AC is blocked by KN-62, which is an inhibitor
of CaM kinases. Furthermore, III-AC is inhibited by coexpression of
III-AC and constitutively activated CaM kinase-II in HEK-293 cells. The
CaM kinase construct used was under the control of the metallothionein
promoter, which allowed the induction CaM kinase-II expression with
Zn
. Ca
inhibition of III-AC in
vivo provides a feedback mechanism for attenuation of
hormone-stimulated adenylyl cyclase activity. Since activation of PKA
can increase Ca
and hormone
stimulation of III-AC is inhibited by Ca
, one might
expect Ca
oscillations to be generated by hormone
stimulation of III-AC. Here, we report glucagon and isoproterenol
stimulation of Ca
oscillations in HEK-293 cells
expressing III-AC and the glucagon receptor.
HEK-293 cells stably expressing
the glucagon receptor (293-G), the glucagon receptor with I-AC
(I-AC-G), or III-AC (III-AC-G) were treated with 100 nM glucagon and individual cells were Ca imaged
using Fura-2 (Fig. 1). In agreement with the observations of
Jelinek et al.(1993), treatment of 293-G cells with glucagon
caused a single spike of intracellular Ca
(Fig. 1A). Additional exposures to glucagon
resulted in no further increase in Ca
. Cells
expressing I-AC and the glucagon receptor gave a similar response; a
single peak of Ca
with no additional increase with
subsequent exposures to glucagon (Fig. 1B). In
contrast, Ca
oscillations were generated when
III-AC-G cells were treated with glucagon (Fig. 1C).
These oscillations were dependent upon the continued presence of
glucagon and were not generated by transient exposure to the hormone.
Figure 1:
Glucagon
stimulation of Ca oscillations in HEK-293 cells
expressing the glucagon receptor and III-AC. HEK-293 cells expressing
the rat glucagon receptor (293-G), or the glucagon receptor with I-AC
(I-AC-G) or III-AC (III-AC-G) were treated with 100 nM
glucagon and Ca
imaged using Fura-2 as described
under ``Experimental Procedures.'' Representative traces from
individual cells are presented.
Glucagon elicited three general types of Ca response in these cells (Fig. 2, Table 1). Only 7%
of III-AC-G cells gave a single Ca
spike (Fig. 2A), 9% showed an intermediate response best
described as spike-plateau (Fig. 2B), and 84% exhibited
Ca
oscillations (Fig. 1C). In
contrast, only 4% of the control 293-G cells responded with
Ca
oscillations (Table 1). The initial
Ca
spike in III-AC-G cells averaged 500 ± 136
nM and was followed by multiple Ca
transients (336 ± 128 nM), which continued for at
least 60 min. These oscillations were at an average frequency of 4.3
peaks/15 min. In 293-G cells, the single Ca
spike
averaged 332 ± 75 nM (Table 1). Several different
stable cell lines expressing III-AC and the glucagon receptor were
examined with analogous results.
Figure 2:
Representative Ca
responses to glucagon in III-AC-G cells. Typical examples of
Ca
responses in III-AC-G cells
treated with 100 nM glucagon are presented. A, 7% of
the cells showed one Ca
spike; B, 8% showed
a spike plateau; C, 85% showed Ca
oscillations. Intracellular Ca
in individual
cells was measured with Fura-2 as described under ``Experimental
Procedures.'' Representative traces from individual cells are
presented.
Figure 3:
Isoproterenol stimulation of
Ca oscillations in HEK-293 cells expressing III-AC. A, HEK-293 cells expressing the glucagon receptor (293-G) or
the glucagon receptor with III-AC (III-AC-G) were treated with 10
µM isoproterenol and Ca
imaged using
Fura-2 as described under ``Experimental Procedures.''
Representative traces from individual cells are
presented.
Figure 4:
Forskolin stimulation of Ca oscillations in HEK-293 cells expressing III-AC. HEK-293 cells
expressing type III adenylyl cyclase (III-AC-G) were treated with 100
µM forskolin and Ca
imaged using Fura-2
as described under ``Experimental Procedures.'' A
representative trace from an individual cell is
presented.
Figure 7:
Stimulation of Ca
oscillations in 293-G cells by combinations of glucagon and IBMX. 293-G
cells were exposed to either 100 nM glucagon (A and B) or 100 µM forskolin (C and D) in the presence (B and D) or absence (A and C) of 300 µM IBMX. Changes in
intracellular Ca
are represented by the relative
change in fluorescence ratio (340 nm/380 nm), which is proportional to
intracellular free Ca
as described under
``Experimental Procedures.'' When present, IBMX (300
µM) was present through the duration of the experiment.
Representative traces from individual cells are
presented.
Figure 5:
Hormone-stimulated cAMP increases in
293-G, I-AC-G, and III-AC-G cells. HEK-293 cells expressing the
glucagon receptor alone (293-G, ) or the glucagon receptor with
I-AC (I-AC-G, &cjs2112;) or III-AC (III-AC-G,
) were treated with
100 nM glucagon (A) or 10 µM isoproterenol (B). Relative cAMP accumulations were
determined as described under ``Experimental Procedures.''
The data are the mean ± S.D. of triplicate
assays.
Figure 6: Forskolin-stimulated cAMP increases in 293-G and III-AC-G cells. HEK-293 cells expressing the glucagon receptor alone (293-G) or the glucagon receptor and III-AC (III-AC-G) were treated with increasing concentrations of forskolin. Relative cAMP accumulations were determined as described under ``Experimental Procedures.'' The data are the mean ± S.D. of triplicate assays.
If Ca oscillations in HEK-293 cells require a threshold cAMP increase,
then it might be possible to stimulate Ca
oscillations in 293-G cells using glucagon or forskolin in
combination with cAMP phosphodiesterase inhibitors, which increase cAMP
signals. The cAMP increases produced in III-AC-G cells by 100 nM glucagon or 10 µM isoproterenol are comparable to
those produced in 293-G cells by a combination of IBMX and glucagon.
Exposure of 293-G cells to either glucagon or forskolin in the absence
of IBMX, a phosphodiesterase inhibitor, produced a single
Ca
peak (Fig. 7, A and C).
IBMX alone had no effect on intracellular Ca
(data
not shown); however, combinations of glucagon and IBMX (Fig. 7B) or forskolin and IBMX (Fig. 7D) resulted in Ca
oscillations. The predominant form(s) of endogenous adenylyl
cyclase in HEK-293 cells is Ca
-inhibitable. For
example, isoproterenol stimulated adenyly cyclase activity in HEK-293
control cells approximately 6.0-fold, and this stimulation was
inhibited 60% by increasing intracellular free
Ca
. These data are consistent with the
proposal that a minimal cAMP increase is necessary for Ca
oscillations.
Figure 8:
Effect of dibutyryl cAMP and (S)-cAMP on intracellular free Ca
in HEK-293 cells. The effect of 1 mM dibutyryl cAMP (A) or 400 µM (S
)-cAMP (B) on intracellular free Ca
in III-AC-G
cells was determined as described under ``Experimental
Procedures.'' Representative traces from individual cells are
presented.
Figure 9:
Inhibitors of cAMP-dependent protein
kinase block hormone-stimulated Ca oscillations in
III-AC-G cells. Intracellular Ca
responses to 100
nM glucagon or 10 µM isoproterenol were monitored
in the presence of 20 µM H-89 (A) or 300
µM (R
)-cAMP (B). The protein
kinase inhibitors were added 30 min prior to imaging. Intracellular
free Ca
was measured and determined as described
under ``Experimental Procedures.'' Representative traces from
individual cells are presented.
KN-62 had no effect on either
basal or carbachol-stimulated intracellular free Ca (data not shown). Although KN-62 did not block the initial
Ca
peak stimulated by glucagon and forskolin,
Ca
oscillations were inhibited by KN-62 (Fig. 10). However, Ca
did
increase approximately 2-fold over the base line and stayed at this
level for at least 30 min. These data are consistent with the
hypothesis that Ca
inhibition of III-AC, by CaM
kinases, may contribute to Ca
oscillations. Because
CaM kinases regulate a number of proteins involved in the regulation of
intracellular Ca
, they may also be important for the
resequestration of intracellular Ca
. For example,
phospholambin (Xu et al., 1993) and a sarcoplasmic reticulum
Ca
pump (Hawkins et al., 1994) are both
phosphorylated by CaM kinases. Phosphorylation of the sarcoplasmic
reticulum Ca
ATPase results in a 2-fold increase in
catalytic activity. Therefore, inhibition of CaM kinase activity in
HEK-293 cells may inhibit the cell's ability to return
intracellular free Ca
to basal levels.
Figure 10:
Hormone-stimulated Ca
oscillations in III-AC-G cells are blocked by the CaM kinase inhibitor
KN-62. Intracellular Ca
responses to 100 nM glucagon or 10 µM isoproterenol were monitored in the
presence of 10 µM KN-62, an inhibitor of CaM kinases.
KN-62 was added 30 min prior to Ca
imaging.
Intracellular free Ca
was measured and determined as
described under ``Experimental Procedures.'' Representative
traces from individual cells are presented.
Figure 11: Effect of adenylyl cyclase activators on phosphoinositol turnover in III-AC-G cells. III-AC-G or 293-G cells were incubated with 10 µM isoproterenol, 100 µM forskolin, 100 nM glucagon, or 1 mM carbachol for 30 min. Total intracellular phosphoinositol was then determined as described under ``Experimental Procedures.'' The data are the mean ± S.D. of triplicate assays.
Figure 12:
Extracellular Ca is not
required for hormone-stimulated Ca
oscillations in
III-AC-G cells. A, the effect of glucagon on intracellular
free Ca
in the absence of extracellular
Ca
(no extracellular Ca
, 1 mM EGTA) was monitored. B, the effect of thapsigargin on
glucagon-stimulated Ca
oscillations was examined.
III-AC-G cells were pretreated with 100 nM thapsigargin in the
absence of extracellular Ca
(Ca
-free, 1 mM EGTA), followed by 100
nM glucagon as indicated. Intracellular free Ca
was measured and determined as described under
``Experimental Procedures.'' Representative traces from
individual cells are presented.
If the primary
source of Ca for oscillations is an internal
Ca
pool, then thapsigargin should inhibit
glucagon-stimulated Ca
oscillations since this drug
is an inhibitor of the intracellular sarcoenodplasmic reticulum
Ca
ATPases (Thastrup et al., 1990; Lytton et al., 1991). Treatment of III-AC-G cells with thapsigargin
in Ca
-free medium caused a rapid release and
depletion of intracellular Ca
stores, and
intracellular free Ca
returned to basal levels within
15 min (Fig. 12B). Glucagon-stimulated Ca
oscillations were completely blocked by pretreatment with
thapsigargin, suggesting that Ca
oscillations were
dependent upon intracellular Ca
pools.
Two of the
major intracellular Ca pools are the ryanodine- and
IP
- sensitive pools, both of which are regulated by cAMP
through PKA (Bird et al. 1993; Nakade et al., 1994;
Yoshida et al., 1992). High concentrations of ryanodine
inhibits the release of Ca
from the
ryanodine-sensitive Ca
pool. Treatment of III-AC-G
cells with ryanodine (1-50 µM) had no effect on
glucagon-stimulated Ca
oscillations in III-AC-G
cells, indicating that the ryanodine-sensitive Ca
pool does not contribute to this phenomenon (Fig. 13).
Figure 13:
Ryanodine does not affect
glucagon-stimulated Ca oscillations in III-AC-G
cells. III-AC-G cells were pretreated with 40 µM ryanodine
followed by 100 nM glucagon as indicated, and intracellular
free Ca
was measured and determined as described
under ``Experimental Procedures.'' A representative trace
from an individual cell is presented.
HEK-293 cells express muscarinic receptors, which are coupled to
mobilization of intracellular free Ca through the
phospholipase C/IP
pathway. The muscarinic agonist
carbachol increases IP
turnover and intracellular
Ca
in these cells. Furthermore, PKA phosphorylation
of IP
receptors stimulates Ca
release
from intracellular stores (Burgess et al., 1991; Bird et
al., 1993; Joseph and Ryan, 1993; Nakade et al., 1994)
and could account for the cAMP-generated Ca
transients caused by forskolin, glucagon, or isoproterenol in
III-AC-G cells. If the IP
-sensitive Ca
pool contributes to the Ca
oscillations
stimulated by forskolin, then pretreatment of III-AC-G cells with
carbachol in the absence of extracellular Ca
should
exhaust the IP
-sensitive pool and inhibit
forskolin-stimulated Ca
oscillations. Incubation of
III-AC-G cells with carbachol in Ca
-free media gave a
single Ca
transient, and subsequent addition of
forskolin did not stimulate Ca
oscillations (Fig. 14). Furthermore, pretreatment of these cells with
forskolin for 5 min, in the absence of external Ca
,
diminished the Ca
increase caused by subsequent
application of carbachol, indicating that both reagents stimulated
Ca
release from a common pool. Collectively, these
data suggest that the major Ca
pool contributing to
glucagon and forskolin-stimulated Ca
oscillations was
the IP
-sensitive pool.
Figure 14:
Carbachol pretreatment inhibits
forskolin-stimulated Ca oscillations in III-AC-G
cells. A, III-AC-G cells were pretreated with 1 mM carbachol and no extracellular Ca
followed by
100 µM forskolin as indicated, and intracellular free
Ca
was measured. B, III-AC-G cells were
pretreated with 100 µM forskolin followed by 1 mM carbachol as indicated, and intracellular free Ca
was measured. Intracellular free Ca
was
measured and determined as described under ``Experimental
Procedures.'' Representative traces from individual cells are
presented.
There is increasing interest in molecular mechanisms for
generation of Ca oscillations in non-excitable cells
(reviewed by Fewtrell(1993) and Berridge(1990, 1992)). Presumably
Ca
oscillations provide enhanced Ca
signals averaged over an extended period of time without the
toxicity associated with persistently elevated
Ca
. One of the most extensively
characterized mechanism for generation of Ca
oscillations is through stimulation of the phospholipase
C/IP
pathway. For example, Bird et al.(1993) have
proposed that IP
-generated sinusoidal oscillations in
intracellular Ca
require negative feedback regulation
of phospholipase C by protein kinase C. In this study we describe a new
mechanism for generation of Ca
oscillations that is
based upon the unique regulatory features of III-AC, an enzyme that is
stimulated by G
-coupled receptors in vivo but
inhibited by elevated Ca
.
Forskolin,
glucagon, and isoproterenol stimulated Ca oscillations in HEK-293 cells that were stably transfected with
III-AC. Control HEK-293 cells did not show Ca
oscillations unless glucagon or forskolin were applied with IBMX.
Since HEK-293 cells express III-AC activity (Xia et al., 1993)
and hormone stimulation of endogenous adenylyl cyclase activity is also
Ca
-inhibitable, it seems likely that this enzyme
contributed to Ca
oscillations in 293-G cells when
endogenous cAMP phosphodiesterase activity was inhibited by IBMX.
Hormone-stimulated Ca
oscillations in III-AC-G cells
were dependent upon PKA activity, the IP
-sensitive
Ca
pool, and they were inhibited by KN-62, a CaM
kinase inhibitor. High levels of cAMP analogues, that were sufficient
to generation single Ca
peaks, did not stimulate
Ca
oscillations suggesting that elevated cAMP was
necessary but not sufficient to account for Ca
oscillations. Glucagon, forskolin and isoproterenol did not
generate Ca
oscillations by stimulating IP
turnover.
What is the mechanism for hormone-stimulated
Ca oscillations in III-AC-G cells? Our data are most
consistent with the following model (Fig. 15). When III-AC is
activated by hormones, cAMP stimulates PKA, which phosphorylates and
activates IP
receptors. As intracellular Ca
rises, III-AC activity is attenuated by CaM kinase(s) and
intracellular cAMP levels decrease because of cAMP phosphodiesterases.
When cAMP levels drops below a threshold point and the IP
receptor is dephosphorylated, Ca
is
resequestered and the cycle can be repeated if III-AC is chronically
exposed to an activator such as forskolin or glucagon. In fact,
Ca
oscillations do not occur with a single exposure
to the hormone or forskolin; the adenylyl cyclase activator has to be
constantly present for the Ca
oscillations to
persist. Interestingly, it has been hypothesized that feedback
inhibition of adenylyl cyclase activity by intracellular Ca
may lead to Ca
oscillations (Rapp and Berridge,
1977; Cooper et al., 1995). The data described in this report
are the first evidence that Ca
inhibition of adenylyl
cyclase activity can lead to Ca
oscillations in
animal cells.
Figure 15:
Mechanism for hormone-stimulated
Ca oscillations in III-AC-G cells. It is hypothesized
that stimulation of III-AC-G by hormones or forskolin leads to
activation of PKA, stimulation of IP
receptors, and
increases in intracellular Ca
. As intracellular
Ca
increases, III-AC activity is inhibited and cAMP
levels are decreased by cAMP phosphodiesterases. When cAMP drops below
a threshold level, Ca
is resequestered and the cycle
is repeated as long as activators of III-AC are present. R
, adenylyl cyclase stimulatory receptor; III-AC, type III adenylyl cyclase; CaM, calmodulin; PKC, protein kinase C; PKA, cAMP-dependent protein
kinase; PLC, phospholipase C; DAG, diacylglycerol; IP
, inositol 1,4,5-trisphosphate; IP
R, IP
receptor/channel; CaMK II/IV, CaM kinase type II or IV; PDE, cAMP
phosphodiesterase.