(Received for publication, July 13, 1995; and in revised form, August 31, 1995)
From the
The effects of immunosuppressant blockers of calcineurin
(protein phosphatase 2B) on cAMP formation and hormone release were
investigated in mouse pituitary tumor (AtT20) cells. Immunosuppressants
enhanced corticotropin-releasing factor- and isoproterenol-evoked cAMP
production in proportion with their potency to block calcineurin.
Further analysis of cAMP production revealed that intracellular
Ca derived through voltage-regulated calcium channels
reduces cAMP formation induced by corticotropin releasing-factor or
-adrenergic stimulation and that this effect of
Ca
is inhibited by blockers of calcineurin. AtT20
cells were found to express at least three species of adenylyl cyclase
mRNA-encoding types 1 and 6 as well as a novel isotype, which appeared
to be the predominant species. In two cell lines expressing very low or
undetectable levels of the novel cyclase mRNA (NCB20 and HEK293 cells
respectively), corticotropin-releasing factor-induced cAMP formation
was not altered upon blockage of calcineurin activity. These data
identify calcineurin as a Ca
sensor that mediates the
negative feedback effect of intracellular Ca
on
receptor-stimulated cAMP production. Furthermore, the effect of
calcineurin on cAMP synthesis appears to be associated with the
expression of a novel adenylyl cyclase isotype, which is highly
abundant in AtT20 cells.
Calcineurin (protein phosphatase 2B) is a
Ca/calmodulin-regulated protein phosphatase first
discovered in brain, where it is highly abundant (0.5-1% of total
protein)(1) . Elucidation of the physiological role of this
protein phosphatase has been relatively slow due to the lack of
specific inhibitors of its enzymatic activity. It is now well
established that the major immunosuppressant compounds cyclosporin A
and FK506 are potent and, with appropriate controls, specific blockers
of calcineurin in leukocytes (2) and other
systems(3, 4) . This observation has led to the
discovery that calcineurin is an essential element of the signal
transduction pathway activated by the T-cell
receptor(5, 6) .
In excitable cells, the functions
of calcineurin are less well understood. Calcineurin has been
implicated in the control of voltage regulated ion channel
activity(7) , particularly with respect to L-type
calcium channels(8) . More recent studies applying
immunosuppressant blockers of calcineurin have shown that the synaptic
vesicular protein dynamin, which is thought to participate in synaptic
vesicle recycling in nerve endings, is a prominent substrate for
calcineurin (9) and that blockage of calcineurin enhances
glutamate release by synaptosomes prepared from rat brain(10) .
In pituitary corticotrope tumor (AtT20) cells (4, 11) immunosuppressants block calcineurin activity
and stimulate Ca-dependent hormone release in
correlation with their calcineurin blocking potency. In hippocampal
brain slices, calcineurin is involved in the induction of long-term
synaptic depression(12) . Finally, ligand-operated ion channels
such as the NMDA receptor (13) or the 5HT
receptor (14) are desensitized by calcineurin.
Taken together, these
data indicate multiple roles for calcineurin in diverse signal
transduction cascades of excitable cells. A common feature of all of
these proposed functions is the Ca-dependent
inhibition of cellular activation. This is the opposite of what has
been observed in nonexcitable cells, such as lymphocytes (5, 6) and adrenocortical glomerulosa
cells(15) , where calcineurin is an intracellular mediator of
the action of stimulatory agents.
The effects of immunosuppressants
on the cAMP signaling system in excitable cells have not been
previously examined. cAMP is a cardinal signaling molecule in pituitary
corticotropes(16) , where its synthesis is activated by
41-amino acid residue CRF()(17) . Increased levels
of cAMP augment intracellular free Ca
concentration
([Ca
]
)(4) ; in
turn, Ca
synergizes with cAMP to trigger the release
of adrenocorticotropic hormone (ACTH)(18) . As
[Ca
]
is also known to
inhibit cAMP formation in several systems (19) and because
immunosuppressants enhanced CRF-induced ACTH release in AtT20
cells(20) , we have examined the effects of immunosuppressants
on CRF-induced cAMP production. The results indicate that in AtT20
cells, calcineurin inhibits CRF-induced cAMP formation and that this is
associated with the expression of a novel isotype of adenylyl cyclase.
For measurements of ACTH release, cAMP production, or
calcineurin activity, the cells were plated on 24-well tissue culture
plates (5 10
cells/well) and used 4-6 days
afterwards. ACTH (21) and cAMP (22) were measured by
specific radioimmunoassays. Calcineurin protein phosphatase activity
was determined by the
P-labeled casein assay (23) or the RII phosphopeptide assay (24) adapted to
measure calcineurin phosphatase activity in AtT20 cell extracts as
described previously(4) .
In the absence of phosphodiesterase blockers, agonist-induced changes of total cAMP content were undetectable at 24 °C. In the presence of IBMX, total cAMP content (cells + medium) increased with time up to 20 min after the addition of CRF and remained constant for up to 30 min. In contrast, intracellular cAMP content peaked between 2 and 5 min and subsequently declined to basal levels even in the presence of the phosphodiesterase blockers. Hence, after establishing that immunosuppressant drugs had the same effect on peak cellular and total cAMP content under these conditions, all experiments shown here report total cAMP content.
In some experiments, nifedipine was given alone during
preincubation to achieve blockage of Ca entry via L
channels; alternatively, the intracellular calcium chelator BAPTA-AM
(20 µM) was used in the preincubation medium in order to
attenuate the rise of intracellular free calcium levels caused by CRF.
Ribonuclease protection assays were performed using an RPA II
kit (Ambion, AMS Biotechnology, Witney, Oxon, UK) according to the
manufacturer's instructions. Briefly, 10 µg of total RNA was
hybridized overnight at 45 °C to 10 cpm of radiolabeled
JP114 antisense riboprobe. Following hybridization, reactions were
digested with single strand-specific RNase, and protected fragments
were resolved on a 6% denaturing polyacrylamide gel, which was fixed
for 30 min in 15% methanol, 5% acetic acid, dried, and exposed to
autoradiographic film at -70 °C.
Figure 1:
Effect of FK506, cyclosporin A, and
MeVal-cyclosporin A (SDZ 202-384) on cAMP
accumulation induced by 10 nM CRF in AtT20 cells in the
presence of 0.5 mM IBMX. Basal cAMP production was 0.6
± 0.08 pmol/well. Data are means ± S.E. and are expressed
as percentage of the increment caused by 10 nM CRF, which was
6.1 pmol/well. Immunosuppressants and IBMX were applied as pretreatment
for 30 min at 37 °C, after which stimulation with CRF was carried
out at 24 °C for 10 min.
Figure 2:
A, effect of cyclosporin A on the time
course of basal and CRF-induced cAMP formation. Data are from a
representative of two experiments. Means ± S.E. (CRF +
vehicle (), CRF + 1 µM cyclosporin A (
),
vehicle (
), cyclosporin A (
)), 0.5 mM IBMX, 0.1
mM rolipram, and cyclosporin A were given as pretreatment for
30 min at 37 °C before the application of 3 nM CRF for 10
min at 24 °C. Data are means ± S.E. *, p < 0.05
compared with respective control group (one-way ANOVA followed by
orthogonal contrasts). B, dependence of the effect of 1
µmol/liter FK506 on the concentration of CRF in the presence of 0.5
mM IBMX. FK506 and IBMX were given as described in A.
*, p < 0.05 compared with the respective vehicle treated
group (one-way ANOVA followed by orthogonal contrasts), n = 4-6/group.
The effect of FK506 on CRF-induced cAMP production could be antagonized by the nonimmunosuppressant analogue L685,818(30) , which also blocked the inhibitory effect of FK506 on calcineurin-mediated dephosphorylation of phosphocasein (Fig. 3, A and B).
Figure 3:
A, the effect of 10 µM L-685,818 on the enhancement of CRF-induced cAMP formation by
FK506. Data are means ± S.E.; the cAMP level in the presence of
10 nM CRF taken as 100% was 12 pmol/well10 min; basal
levels were 1.6 pmol/well
10 min. B, the effect of
L-685,818 on the inhibition of calcineurin activity by 100 nM FK506 in AtT20 cells. Cells were preincubated in 24-well plates
for 30 min at 37 °C with FK506 ± the indicated
concentrations of L-685,818. Subsequently cell extracts were prepared
by hypoosmotic lysis, and calcineurin activity was measured by the
phosphocasein method. The activity measured in the absence of FK506 was
1.4 nmol mg of protein
min
and
was taken as 100%. Data are means ± S.E., n =
3/group.
Furthermore, data reported elsewhere (20) showed that the
addition of graded amounts of Ca with CRF to cells
depleted of Ca
and pretreated with the ionophore
A23187 produced a concentration-dependent inhibition of CRF-induced
cAMP production to levels seen in nondepleted cells incubated in medium
containing 2 mM Ca
. The effect of exogenous
Ca
could be inhibited by FK506, which failed to alter
cAMP accumulation in the absence of Ca
(20) .
Figure 4: Effect of pertussis toxin (PTX, 1 µg/ml for 18 h) on the modulation of CRF-induced cAMP formation by FK506 and somatostatin (SRIF). AtT20 cells were preincubated with various concentrations of FK506 (Control FK506, PTX FK506) or somatostatin (Control SRIF, PTX SRIF) for 30 min at 37 °C and challenged with 10 nM CRF for 10 min at 24 °C; IBMX (0.5 mM) was present throughout. Data are means ± S.E., n = 6/group.
FK506 had no
significant effect on cAMP accumulation evoked by 10 or 30 µM forskolin, a drug that at these concentrations activates adenylyl
cyclase independent of G. Loading of the cells with
BAPTA-AM caused a small (15%), statistically significant (p < 0.05) enhancement of forskolin-evoked cAMP accumulation (not
shown) .
Finally, in contrast to the effects of FK506 and cyclosporin A, pretreatment with other blockers of protein phosphatases such as calyculin A (1-30 nM) and okadaic acid (0.2-5 µM), caused a concentration-dependent inhibition (up to 80%) of CRF-induced cAMP accumulation (not shown and (36) ).
Figure 5:
Effect of FK506 (0.5 µM) on
basal and CRF-induced ACTH secretion in AtT20 cells (A) and
antagonism of the effect of FK506 (0.5 µM) on CRF-induced
ACTH release by L685,818, which had no effect on basal ACTH secretion
in this system even at 5 µM (B). n = 4/group, means ± S.E. In panel A, the
values for basal and CRF-stimulated ACTH release taken as 100% were 15
± 1 and 22 ± 1 fmol/well30 min, respectively. *, p < 0.05 when compared with the group not receiving
L,685,818, one-way ANOVA followed by orthogonal contrasts. Cells were
pretreated with immunosuppressant analogues (open bars, L685,
818; shaded bars, L685, 818, and FK506) for 30 min at 37
°C, and then the medium was changed and the cells were challenged
with 10 nM CRF at 24 °C for 30
min.
Figure 6:
Comparison of the sequence amplified from
AtT20 cell RNA using primer set B with the corresponding sequences of
mammalian adenylyl cyclases found in current data bases (EMBL, GenBank,
SwissProt). The numbers relate to the amino acid sequence of rabbit
ACtype5 (ocmradcyv). The alignment was generated by the PileUp
program of the GCG package. The sequence is annotated by ()
showing amino acid identity of the novel sequence with at least one
previously reported AC, (
) shows functionally conservative
substitutions(-) denotes nonconservative substitutions as defined
by Krupinski et al.(52) . The abbreviations used are
as follows: Hum9, Human AC type9 (GenBank #D25538); mmu12919, mouse AC type7; cya2_rat, rat
ACtype2; cya_rat, rat ACtype4; hsadencyr8,
Human ACtype8; ratacviii, rat ACtype8; a46187, human
ACtype5; cya6_mouse, mouse ACtype6; a49201,
mouse ACtype5; cya6_rat, rat ACtype6; cya6_canfa, dog ACtype6; s29717, rat
ACtype5; ocmradcyv, rabbit ACtype5; cya5_canfa, dog ACtype5; cya1_bovin, bovine Actype1; cya3_rat, rat ACtype3; jp114, AtT20 new
sequence(mouse ACtype10)
Northern blot analysis of total RNA using the novel adenylyl cyclase 180-bp cDNA fragment as a probe indicated hybridization to an approximately 9-kilobase mRNA expressed in AtT20 cells (Fig. 7), and a single hybridizing species of RNA of similar size was detected in NCB20 and HEK293 cells at much lower intensity (Relative hybridization intensity of 9-kb band (arbitrary units): AtT20, 0.38; NCB20, 0.03; HEK293, 0.07).
Figure 7:
Detection of novel adenylyl cyclase mRNA
in various cell lines. Left panel, Northern analysis of AtT20
cell total RNA; note approximately 9-kb RNA species hybridizing with P-dCTP-labeled JP114 cDNA (arrow). Right
panel, ribonuclease protection assay of using JP114 cRNA: RNA from
AtT20 (lanes 1 and 2), COS7 (lanes 3 and 4), HEK293 (lanes 5 and 6), and NCB20 (lanes 7 and 8) cells. Lane 9 contained
yeast RNA as negative control; lane 10 contained undigested
probe. Note abundant, approximately 160 bp protected RNA species in
AtT20 cells and much less intense band in NCB20
cells.
As a potentially more sensitive alternative, mRNA expression
was also assayed by ribonuclease protection using a radiolabeled
antisense riboprobe transcribed from the novel adenylyl cyclase cDNA.
An approximately 160-bp ribonuclease-protected RNA species (Fig. 7) indicates that the novel adenylyl cyclase mRNA is
highly abundant in AtT20 cells, whereas much lower levels are present
in NCB20 cells, and in HEK293 cells the mRNA was undetectable. ()
Calcineurin protein phosphatase activity (substrate RII subunit peptide (24) ) in cell extracts prepared from AtT20, HEK293, and NCB20 cells fell to 25, 19, and 42% of the respective control activities after pretreatment with 1 µM FK506. Similar to AtT20 cells, CRF-stimulated cAMP formation in HEK293 cells (Fig. 8) as well as NCB20 cells (not shown), and this was enhanced by the depletion of intracellular calcium stores as described for AtT20 cells. However, while in AtT20 cells FK506 consistently enhanced CRF-induced cAMP formation, in NCB20 cells only one out of four experiments gave a statistically significant enhancing effect of 1 µM FK506 on CRF-induced cAMP accumulation, and in none out four experiments in the case of HEK293 cells (Fig. 8). No effects of cyclosporin A were found in either system (not shown).
Figure 8:
Stimulation of cAMP accumulation in HEK293
cells by corticotropin-releasing factor. Cells were pretreated with the
calcium depleting medium at 37 °C in the presence of 1 mM IBMX plus or minus 1 µM FK506 for 30 min before the
addition of CRF for 10 min at 24 °C. In the case of the control and
the FK506-treated group, the CRF solution contained sufficient
CaCl in buffered EGTA to bring the medium to nominally 2
mM free extracellular Ca
. Unstimulated cAMP
content was 0.05 ± 0.006 pmol/well and was unaltered by
depletion of calcium or preincubation with FK506. n =
4/group; data are means ± S.E.; *, p < 0.01 when
compared with the control group in 1-way ANOVA followed by orthogonal
contrasts.
These data show that receptor-stimulated cAMP formation may be inhibited by calcineurin and that this regulation is associated with the expression of a novel adenylyl cyclase mRNA.
All studies of cAMP formation reported here were carried out in the presence of blockers of phosphodiesterase, and hence the effects observed relate to changes in the rate of synthesis of cAMP rather than to its degradation.
Evidence for the involvement of calcineurin in the control of cAMP
accumulation is provided by the use of immunosuppressant compounds
previously (4) shown to block calcineurin activity in AtT20
cells with the same order of potency that they influenced cAMP
accumulation (present study). The EC for FK506 to block
calcineurin activity is considerably higher in AtT20 cells (
10
nM) than in T lymphocytes (
0.8 nM), which is
probably attributable to differences in the respective cellular levels
of calcineurin and FKBP12 in these systems. Importantly, L685,818, an
analogue of FK506 (30) that binds to the prolylisomerase
FKBP-12 in a manner similar to FK506 but does not give rise to a
drug-protein complex that inhibits the activity of calcineurin,
reversed the effects of FK506 on calcineurin activity and cAMP
formation as well as ACTH release. When given alone, L685,818 had no
discernible effect on cAMP formation or ACTH secretion, further
suggesting that the changes observed upon treatment with FK506 are due
to the inhibition of calcineurin and not due to the blockage of the
prolylisomerase activity of FKBP12. Finally, neither FK506 nor
cyclosporin A were effective in cells deprived of
Ca
(20) .
Taken together, these characteristics justify the conclusion that the effects of immunosuppressants described here are attributable to the inhibition of calcineurin.
The production of cAMP in AtT20 cells is under
inhibitory control by [Ca]
.
Stimulation with cAMP is known to elicit a rise of
[Ca
]
in these cells, which is
largely derived from the extracellular pool by influx through
dihydropyridine-sensitive Ca
-channels (26, 27, 28) . Thus the
[Ca
]
signal is a measure of the
electrical activity of the cells and, in addition to triggering hormone
release, provides feedback inhibition to the chemical messenger system
that generates it. In the case of CRF-induced cAMP formation, this
feedback appears to be mediated by calcineurin.
Several
possibilities have to be considered with respect to the site of action
of Ca/calcineurin in the signal transduction cascade.
An action of calcineurin at the receptor level is conceivable; however, the prevailing concept of G-protein-coupled receptors (38) dictates that receptor down-regulation or uncoupling is largely due to the action of protein kinases while protein phosphatases reverse this process. In contrast, the present data implicate calcineurin as an inhibitor of receptor-stimulated cAMP production.
Dephosphorylation of the coupling protein G is also a
possible site of regulation by calcineurin(39) . Once more,
current evidence in the literature associates protein phosphorylation
with down-regulation of G-protein function and implicates protein
phosphatases in the restoration of the cellular
response(40, 41) .
With respect to the effector
enzyme adenylyl cyclase, these proteins have lately emerged as dynamic
sites of signal integration(42) . At least two types of
cyclase, types 5 and 6(43) , are inhibited by
Ca, but the mechanism of this effect has not been
elucidated(33) . The inhibition of type 5 and 6 cyclase by
Ca
is most marked after stimulation by agonists such
as isoproterenol in chick heart cells(44) , or VIP in
GH
C
pituitary tumor cells (45) , but
much less prominent after activation with forskolin in
GH
C
cells(45) . Overall this is
analogous to the observations made here, which in the first instance
suggest a prominent action of calcineurin at or before the level of
G-protein effector coupling. However, as multiple types of adenylyl
cyclase coexist in all cell types analyzed to
date(33, 46, 47) , and forskolin appears to
activate these by different efficacies and mechanisms, an effect of
Ca
on catalytic activity as opposed to the
interaction with the G
-subunit-coupling site cannot
be excluded (43) . Reverse transcriptase PCR analysis and
sequencing of amplified cDNAs clearly show that at least three types of
adenylyl cyclase mRNA (type 1 and 6 as well as a novel isotype) are
co-expressed in AtT20 cells, and thus the above considerations also
apply to this system.
It is unlikely, that type 1 cyclase is
involved in the effects reported here as it is invariably stimulated by
Ca, whereas Ca
was strongly
inhibitory to both CRF and
-adrenergic stimulation of cAMP. Type 6
adenylyl cyclase could be implicated as it is inhibited by
Ca
. However this isotype is abundant in NCB20 (33) as well as HEK293 cells (47) where the stimulation
of cAMP accumulation through endogenously expressed receptors for CRF
was not altered by immunosuppressants, despite a marked inhibition of
calcineurin activity measured using the RII substrate phosphopeptide.
Importantly, the novel adenylyl cyclase homologue mRNA was found in
very low amounts in NCB20 cells and HEK293 cells, whereas it appears to
be the predominant adenylyl cyclase isotype mRNA in AtT20 cells. A
partial mammalian sequence that is identical except for a single amino
acid to the one reported here has been previously designated as
adenylyl cyclase type 10(48) . Results from this laboratory (49) show that the 9-kb mRNA detected in AtT20 cells contains a
full-length adenylyl cyclase coding sequence giving rise to an adenylyl
cyclase inhibited by calcineurin.
A previous study (15) has reported that calcineurin is stimulatory to cAMP formation; immunosuppressants blocked the enhancement of ACTH-evoked cAMP production by angiotensin II and activators of protein kinase C in bovine adrenal cortical cells as well as transfected COS-7 cells. As adenylyl cyclase type 10 mRNA is undetectable in COS-7 cells and protein kinase C activation is inhibitory to cAMP production in AtT20 cells(36) , it is unlikely that adenylyl cyclase type 10 is involved in the enhancement of cAMP production by calcineurin as reported by Baukal and co-workers(15) . Another study, using partially purified solubilized bovine brain adenylyl cyclase (50) reported inhibition of cyclase activity by calcineurin; however, this was attributed to the sequestration of endogenous calmodulin in the enzyme preparation by calcineurin, and the consequent inhibition of a calmodulin-stimulated cyclase activity. Thus, whether calcineurin regulates cyclase type 10 directly or through an intermediary phosphoprotein specific to AtT20 cells remains to be determined by future studies.
Taken together, the present data
indicate that in AtT20 cells calcineurin is a
Ca-operated feedback inhibitor of CRF or
-adrenergic receptor-evoked cAMP responses. As
[Ca
]
is largely derived through
voltage-regulated Ca
channels in AtT20 cells, these
cells exemplify a case where calcineurin functions as a link between
the cAMP-generating and electrical signaling systems of the cell. The
potential functional significance of this mechanism is illustrated by
changes of hormone secretion that parallel the enhancement of the cAMP
signal. Immunosuppressants augmented CRF-induced ACTH secretion and
attenuated the inhibitory effect of adrenal
corticosteroids(20) . Our findings conform with previous
reports (7, 10, 13, 14) in showing
that calcineurin is a fundamental negative feedback regulator of
cellular responses in excitable cells and extend this function to the
cAMP signal transduction cascade. In this latter respect the data also
support the earlier notion(51) , that calcineurin is a generic
antagonist of cAMP-induced stimulatory mechanisms.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) MMU30602[GenBank], Z50190[GenBank], and Z46958[GenBank].
Addendum-The cDNA sequence of the novel adenylyl cyclase cloned from AtT20 cells (now called type 9) has been deposited in EMBL/GenBank by two groups under accession numbers MMU30602 and Z50190. A further highly homologous sequence from Xenopus laevis is found under accession number Z46958