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(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
Calcineurin (protein phosphatase 2B) is a
Ca 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 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 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(
For measurements of ACTH release, cAMP production, or
calcineurin activity, the cells were plated on 24-well tissue culture
plates (5
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
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
Figure 1:
Effect of FK506, cyclosporin A, and
MeVal
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 (
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/well
Furthermore, data reported elsewhere (20) showed that the
addition of graded amounts of Ca
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 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/well
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 (
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
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
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 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 Several
possibilities have to be considered with respect to the site of action
of Ca 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 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 It is unlikely, that type 1 cyclase is
involved in the effects reported here as it is invariably stimulated by
Ca 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
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
Volume 270,
Number 47,
Issue of November 24, 1995 pp. 28055-28061
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
/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) .
-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.-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.
)(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.
Cell Culture
AtT20 D16:16 mouse anterior
pituitary tumor cells were maintained in culture as described
previously(21) . NCB20 mouse neuroblastoma 
hamster
brain hybridoma cells (courtesy of Dr. Beth Hoffman, National Institute
of Mental Health, Bethesda, MD) were cultured in 10% newborn calf serum
and Dulbecco's minimal essential medium with
hypoxanthine/aminopterin/thymidine supplement (Life Technologies,
Paisley, Scotland, United Kingdom). Human embryonic kidney (HEK293)
cells (courtesy of Dr. Lorraine Anderson, Medical Research Council
Reproductive Biology Unit, Edinburgh, Scotland, UK) were maintained as
AtT20 cells. 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) .
Measurement of cAMP Responses to Agonists
This
protocol was established using AtT20 cells and was also applied in
experiments with the other two cell lines. Experiments were all carried
out in Hank's balanced salt solution containing 2 mM
CaCl and 1 mM MgSO buffered with 25
mM HEPES, pH 7.4, and supplemented with 0.25% (w/v) of bovine
serum albumin. The cells were preincubated in serum-free medium for 1
h, after which fresh medium containing blockers of phosphodiesterase,
0.5-1 mM IBMX, and/or 0.1 mM rolipram along
with various other agents as specified below and in the figure legends
were applied for 30 min at 37 °C. Subsequently, the cells were
cooled to 24 °C (5-10 min in a water bath), and agonists were
added for 10 min. The reaction was stopped by the addition of 0.2 M HCl to achieve a final concentration of 0.1 M(25) .Depletion and Repletion of Intracellular Calcium
Pools
Calcium depletion was initiated during preincubation in
the presence of phosphodiesterase blockers, which were applied as
described in the preceding section. Cells were preincubated for 30 min
in medium containing 2 mM EGTA and no added Ca supplemented with 5 µM A23187 and 2.5
µM nifedipine in order to deplete rapidly mobilized
cellular stores of Ca
and to ensure that L-type
Ca
channels, the principal avenue of
voltage-regulated Ca
influx in AtT20 cells (26, 27, 28) were fully blocked. Thus,
Ca
, added as chloride salt to the extracellular fluid
at the time agonist stimulation was initiated, would enter largely
through the pores made by the ionophore A23187. The rationale for this
pretreatment is that calcineurin reportedly influences L channel
activity(7, 8) , whereas the treatment regimen used
here would make Ca
entry independent of this
regulation.
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.
Pretreatment with
Immunosuppressants
Immunosuppressant analogues (FK506, Fujisawa
Ltd, Osaka, Japan) cyclosporin A, and SDZ 220-384 (MeVal cyclosporin A, a weak agonist) (29) , (Sandoz Pharma,
Basel, Switzerland), L-685,818 an FK506 antagonist, (Merck) were
applied during the 30-min preincubation period, invariably in the
presence of phosphodiesterase blockers, and in some cases to cells
undergoing the calcium depletion protocol described above.
Immunosuppressants were made up in ethanol at 10M and diluted with the incubation medium to the desired
final concentrations. In some cases, cells were preincubated with
L-685,818 a structural analogue of FK506 that binds to FKBP12 and
inhibits prolylisomerase activity but is devoid of immunosuppressant
activity (30) for 10 min before the addition of FK506.
Preincubation was carried out at 37 °C, because immunosuppressants
are taken up into cells through an active transport process involving
the multidrug resistance transporter glycoprotein (31) , and
the uptake into cells is temperature-dependent (32) . Test
incubations with agonists were carried out at 24 °C, because at
this temperature the effects of immunosuppressants on cAMP formation
were more pronounced than at 37 °C. At present, we have not
identified the cause for the temperature-dependent difference in the
efficiency of immunosuppressants to influence cAMP formation.
Secretion of ACTH
Incubations for ACTH secretion
were carried out as described previously (21) except that the
test incubation with CRF was for 30 min at 24 °C, to allow
comparisons with the conditions of cAMP accumulation experiments.
Immunosuppressants were applied as in cAMP experiments; blockers of
phosphodiesterase were not used.Amplification and DNA Sequence Analysis of Adenylyl
Cyclases cDNAs in AtT20 Cells
Total RNA was prepared from
approximately 10
cells using Trizol reagent (Life
Technologies, Inc.) according to the manufacturer's instructions.
Reverse transcriptase PCR was carried out using an RNA PCR kit (Perkin
Elmer). Briefly, 0.8 µg of total RNA was denatured at 95 °C for
5 min and then annealed with 2.5 µM random hexanucleotide
primers for first-strand cDNA synthesis, which was carried out for 15
min at 42 °C in a 20-µl reaction mixture containing 10 mM Tris
HCl, pH 8.3, 50 mM KCl, 5 mM
MgCl, 1 mM dNTP, 20 units of RNase inhibitor, and
50 units of Moloney murine leukemia virus reverse transcriptase. The
reaction was terminated at 99 °C for 5 min and then cooled to 4
°C and stored on ice. PCR was performed using degenerate
oligonucleotides, either pair A or pair B corresponding to highly
conserved regions within the first (pair A,
5`-CTCATCGATGGIGAYTGYTAYTAYTG-3`; 3`-GGCTCGAGCCAIACRTCRTAYTGCCA-5`,
expected product size 220 bp) and second (pair B,
5`-GAAGCTTAARATIAARACIATIGGI(T/A)(C/G)IACITAYATGGC-3`;
3`-GGGATCCACRTTIACIGTRTTICCCCAIATRTCRTA-5`, expected product size 180
bp) cytoplasmic domains of previously cloned mammalian adenylyl
cyclases(33, 34, 35) . For PCR, the reverse
transcription reaction (20 µl) was expanded to 100 µl and
contained 10 mM TrisHCl, pH 8.3, 50 mM KCl, 2
mM MgCl, 200 µM each dNTP, 35 pmol of
each primer, and 2.5 units of Amplitaq DNA polymerase. PCR reactions
were overlaid with mineral oil (Sigma) and denatured at 95 °C for 3
min followed by 5 cycles (60 s denaturation at 94 °C, 60 s
annealing/extension at 45 °C) and then a further 35 cycles (60 s
denaturation at 94 °C, 60 s annealing/extension at 55 °C), and
finally 7 min of annealing/extension at 55 °C. An aliquot (5%) of
each reaction was analyzed by agarose gel electrophoresis (3% Nusieve
(Flowgen). Products within the expected size range for each primer pair
were excised from the gel, purified using a Wizard(TM) PCR Prep kit
(Promega), and ligated into the vector pGEM-T (Promega). Clones
containing an insert of the expected size were identified, and their
DNA sequence was determined by the dideoxynucleotide method (Sequenase
2.0 kit, U. S. Biochemical Corp.). Clone JP114, containing 180 bp of
cyclase sequence was isolated and used for generating cDNA and cRNA
probes for RNA detection.Detection of mRNA Expression
Northern analysis was
performed using standard procedures. Briefly, 10 µg of total RNA
was separated by formaldehyde gel electrophoresis and transferred by
blotting onto positively charged nylon membrane (Appligene) and then
fixed by baking at 80 °C and prehybridized at 42 °C for 2 h in
50% deionized formamide, 5 saline/sodium phosphate/EDTA, 0.5
Denhardt's solution, 0.1% (w/v) SDS, 0.2 mg/ml denatured
salmon sperm carrier DNA, and 10% Dextran sulfate. Random-primed
[P]dCTP-labeled DNA probe (50 ng; >10
cpm/µg) was then added, and hybridization continued overnight
at 42 °C. The membrane was washed twice for 20 min in 2
SSC, 0.1% SDS, followed by 20 min in 1 SSC, 0.1% SDS at 42
°C and finally 20 min in 0.5 SSC, 0.1% SDS at 50 °C
before wrapping in cling-film and exposing to autoradiographic film at
-70 °C or to Molecular Dynamics PhosphorImager cassettes and
quantified with the ImageQuant software using the 28 S RNA band as a
standard for RNA loading. Division of the integrated volume of pixels
of the selected radiolabeled band with the integrated volume of the
internal standard band yields the relative hybridization intensity,
which was used to compare the intensity of labeled RNA bands within
blots.
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.
Enhancement of CRF-stimulated cAMP Production by
Immunosuppressants
Blockers of calcineurin activity enhanced
CRF-induced cAMP production (Fig. 1). This effect was
statistically significant (p < 0.05 or less) at 5-30
min after the addition of CRF (Fig. 2A). An enhancement
of cAMP formation by FK506 was apparent at lower concentrations
(0.1-10 nM) of CRF, while the maximal response was
unchanged (Fig. 2B).
-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.
), 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.
10 min; basal
levels were 1.6 pmol/well10 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.
Receptor-evoked Synthesis of cAMP Is under Inhibitory
Control by Intracellular Ca
Lowering of [Ca and
Calcineurin
]
by a variety of methods all markedly increased the cAMP response
to 10 nM CRF (control, 100 ± 9; Ca depletion protocol, 198 ± 18; nifedipine (0.1
µM) in preincubation, 205 ± 22; BAPTA-AM (20
µM) in preincubation, 275 ± 25). Data are means
± S.E. of the increment over unstimulated cAMP levels and are
expressed as percentage of the control CRF group run in each
experiment, n = 6/group. p < 0.01 for all
treatments when compared with control by one-way ANOVA and
Dunnett's test. (Unstimulated cAMP levels were not affected by
these manipulations of [Ca
]
,
which were all initiated during the 30-min preincubation period). The
effect of BAPTA-AM on CRF-induced cAMP formation was statistically
significant (p < 0.05) by 2 min after the addition of CRF
and at all subsequent time-points studied up to 20 min (not shown). 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) .
Site and Specificity of Immunosuppressant
Action
The effect of FK506 on CRF-induced cAMP formation was
also evident after 16 h of pretreatment of AtT20 cells with pertussis
toxin (1 µg/ml) (Fig. 4), which strongly suppressed
inhibitory G-protein function as assessed by the attenuation of
somatostatin-mediated inhibition of cAMP formation. Pertussis toxin
treatment also had no effect on the suppression of CRF-induced cAMP
formation by extracellular Ca in
Ca
-depleted cells (not shown).
. Loading of the cells with
BAPTA-AM caused a small (15%), statistically significant (p < 0.05) enhancement of forskolin-evoked cAMP accumulation (not
shown) .Enhancement of CRF-stimulated ACTH Release by
FK506
Blockage of calcineurin activity by FK506 enhanced the
release of ACTH evoked by CRF (Fig. 5A), and this
action was prevented by L-685,818 (Fig. 5B). Note, that
the apparent EC values of FK506 to inhibit calcineurin
activity in AtT20 cells(4) , to stimulate ACTH release, and to
augment cAMP accumulation induced by CRF are all approximately 10
nM.
30 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.
Isoproterenol stimulates cAMP production through
-Adrenergic Stimulation Is under Similar Regulation
by Calcineurin-adrenergic receptors in AtT20 cells(37) , and
this was enhanced by both BAPTA-AM and FK506 (Table 1).
Effect of Immunosuppressants on cAMP Accumulation in
AtT20 Cells Correlates with the Expression of a Novel Adenylyl Cyclase
mRNA
In order to determine the profile of adenylyl cyclase
isoforms present in AtT20 cells, two sets of degenerate oligonucleotide
primers were used to analyze AtT20 cell total RNA for adenylyl
cyclase-related sequences by means of reverse transcriptase PCR. Using
primer set B, a PCR product of approximately 180 bp was obtained. DNA
sequence analysis revealed that approximately 8% of the subcloned
180-bp cDNA fragments amplified proved to be identical to type 6
adenylyl cyclase. The majority (>90%), however, gave a novel
sequence that was highly homologous to the primary amino acid sequences
of known mammalian adenylyl cyclases found in current data bases but
was not identical to any of these (Fig. 6). Type 1 adenylyl
cyclase was detected in AtT20 cells using primer set A.
)
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)
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.
)
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.
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) .
]
.
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./calcineurin in the signal transduction cascade.
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) ., 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
GHC 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., 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.
-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.
))
We thank Dr. A. Baukal (NICHD, National Institutes of
Health, Bethesda, MD) for the cAMP antiserum, Dr. H. Fliri (Sandoz
Pharma, Basel, Switzerland) for cyclosporin A and SDZ 220-384,
Dr. N. Sigal (Rahway, NJ) for L-685,818, Dr. G. B. Makara (Budapest)
for the ACTH antiserum, Dr. T. Takaya (Fujisawa Ltd., Osaka, Japan) for
FK506, and Dr. C. B. Klee (National Cancer Institute, Bethesda, MD) for
helpful discussions.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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