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Division of Endocrinology and Metabolism (S.E.M., H.V.), Department of Medical Physiology, The Panum Institute, University of Copenhagen, Blegdamsvej 3C, DK-2200 Copenhagen N, Denmark; and Department of Medical Gastroenterology C (T.S.), Herlev University Hospital, Denmark
Address all correspondence and requests for reprints to: S. E. Mau, Division of Endocrinology and Metabolism, Department of Medical Physiology, The Panum Institute, University of Copenhagen, Blegdamsvej 3C, DK-2200 Copenhagen N, Denmark. E-mail: smau{at}pop.mfi.ku.dk
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, ß, 
, and 
) detectable in lactotroph-enriched
cell cultures derived from peripubertal male rats, whereas both
compounds significantly increased translocation of PKC and ß from
the cytosol to the plasma membrane induced by SP. The and
subspecies were not affected by VIP and forskolin. Moreover, VIP and
forskolin also stimulated SP-induced formation of
Ins(1,4,5)P3 while having no effect on basal
inositol phosphate turnover. The effects of VIP and forskolin on PKC
isozyme distribution could be blocked by pretreating cells with the PKA
inhibitor rp-cAMP. On the other hand, SP potentiated the effect of VIP
and forskolin on cAMP formation while having no effect on the cAMP
pathway when it was not triggered by an appropriate agonist.
Down-regulation of PKC activity by long term 12–0-tetradecanoylphorbol
13-acetate (TPA) treatment (24 h) diminished, but did not abolish, the
effect of SP on VIP-stimulated cAMP production. Staurosporine and
dopamine inhibited the potentiating effect of SP on cAMP accumulation.
TPA, which translocates PKC, ß, and in lactotrophs, had a
synergistic effect on cAMP formation induced by VIP, but did also,
unlike SP, display cAMP rising abilities when cells were not exposed to
VIP and forskolin. Discharging intracellular Ca2+ by
thapsigargin pretreatment had no effect on the basal cAMP concentration
or the VIP-induced cAMP response, whereas exposure of cells to SP,
thapsigargin, and VIP resulted in a decrease of the cAMP response
compared with SP + VIP. The potentiating effect of SP on the VIP
response could also be inhibited, but not blocked, by staurosporine. On
the basis of these results, it is concluded that there exists
substantial cross-talk between the cAMP/PKA and
PKC/Ins(1,4,5)P3 messenger systems in
lactotroph-enriched cell cultures. Key effectors seem to be PKA, one or
more of PKC , ß, and
Ins(1,4,5)P3-sensitive Ca2+
stores. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, ß, 
, , 
and protein kinase C
isozymes came from Life Technologies (Roskilde, Denmark). Biotinylated
swine immunoglobulins to rabbit immunoglobulins and
Strept-ABComplex-HRPR were purchased from Dako A/S
(Copenhagen, Denmark), Percoll from Pharmacia Upjohn (Hillerød,
Denmark) and rp-cAMP from Biolog Life Science Institute (Bremen,
Germany). All other reagents were from Sigma.
Animals
Peripubertal Wistar male rats (42 days, 180 g) were housed
in a temperature- and humidity-controlled room with a 12-h light, 12-h
dark cycle and allowed free access to a standard pellet diet and water.
Handling of the animals was conducted according to the NIH Guide for
the Care and Use of Laboratory Animals and was approved by the
Committee on Animal Care and Use under the Danish Department of
Justice.
Preparation of lactotroph-enriched cell cultures
Rats were gassed with CO2 and decapitated. Dispersed
anterior pituitary cells were obtained by trypsination and mechanical
disruption as described previously (16). Separation of the
heterogeneous pituitary cell population was performed by a
discontinuous Percoll gradient centrifugation (16). An enriched
population of lactotrophs was identified in interphase 1 by
immunocytochemistry as previously described (18). For each cell
separation, the content of lactotrophs in the different cell layers was
determined (average 86 ± 4% in interphase 1 vs.
41 ± 2% in the crude dispersed cell population). The density of
each Percoll layer in the gradient was verified by simultaneous
centrifugation of density markers beads (1.063, 1.077, and 1.085).
Lactotroph-enriched cell suspensions were washed three times in RPMI
1640 and placed in microtiter plates (for PKC measurements, 96
wells/plate, incubation volume 250 µl) or plated in culture dishes
(cAMP measurements, 24 wells/plate, incubation volume 1 ml, or inositol
phosphate experiments, 12 wells/plate, incubation volume 2 ml) at a
concentration of 106 cells/ml in RPMI 1640 supplemented
with 10% (vol/vol) charcoal-extracted FCS. Cells were grown in a
CO2/air incubator at 37 C for 4 days at which time they
were firmly attached to the bottom of the wells.
Subcellular fractionation, extraction, partial purification of PKC
and Western blotting of PKC isozymes
In PKC isozyme-stimulation experiments, cells were washed three
times with RPMI 1640 and substance P, VIP,
12-O-tetradecanoylphorbol 13-acetate (TPA), or forskolin
were diluted in RPMI 1640 + 0.05% (wt/vol) BSA (medium A) and added to
cell cultures. TPA and forskolin were dissolved in dimethyl sulphoxide
(DMSO) and before experiments diluted in medium A. The final
concentration of DMSO in wells never exceeded 0.001% (vol/vol) and did
not affect basal distribution of PKC isozymes, levels of cAMP in
resting cells or secretion of PRL. Vehicle containing the same amount
of DMSO served as control when appropriate. In experiments using
rp-cAMP, rp-cAMP was preincubated for 10 min before adding VIP or
forskolin. The optimal preincubation time and concentration of rp-cAMP
were determined a priori in separate experiments. VIP and
forskolin were applied 5 min before addition of SP, after which
reactions were run for additional 5 min and terminated by aspirating
the medium and adding 250 µl of ice-cold buffer A (Tris-base 20
mM, pH 7.5, EDTA 2 mM, EGTA 0.5 mM,
phenylmethylsulphonyl fluoride, 1 mM, bacitracin 40
µg/ml, leupeptin 10 µg/ml, benzamidine 10 mM and
aprotinin 100 kallikrein inhibiting U/ml). Cells were scraped and
subjected to subcellular fractionation by homogenization and
differential centrifugation (16). The soluble and particulate fractions
were extracted for PKC by Nonidet P 40 and PKC activity was partly
purified by diethylaminoethyl chromatography and SDS-PAGE (16).
Proteins were transferred from gels for 2 h in a semi dry blotter
II (Kem En Tec, Copenhagen, Denmark) at 1–3 V/cm. PKC isozymes were
visualized by polyclonal antibodies and StreptABComplex (16)
Quantification of immunoblots
The densities of the PKC bands were determined by scanning with
a Hamamatsu video camera. The images were numerically quantitated
according to band density by a software package (Image 1.41, Wayne
Rasband, NIH) on a Macintosh IIci computer. The raw numerical data were
divided by the protein concentration of each sample, expressed as
density units per µg protein and subsequently transformed into
percentage change in immunoreactivity (IR) with 100% representing the
amount of IR detected in resting cells.
Protein kinase C assay
To evaluate the catalytic activity of PKC in the particulate
fraction, cells were treated and stimulated exactly as described above.
After partial purification of PKC, the activity in eluate samples of
the particulate fractions was determined by transfer of
[-32P] from [-32P]ATP into a
synthetic peptide MBP4–14 as described in details
previously (16).
Determination of cAMP
Cells were stimulated under exactly the same conditions as
described above in the section dealing with western blotting. SP or TPA
was added 5 min before VIP or forskolin, after which reactions were
continued for 5 min. Extraction and determination of cAMP in cell
cultures were performed by Amersham’s [125I]cAMP RIA kit
according to the instructions of the manufacturer (Amersham DK,
Birkerød, DK). The inter and intraassay variations were 7% and 5%,
respectively.
Determination of [3H]inositol 1,4,5-trisphosphate
Labeling of enriched lactotrophs with
myo-[3H]inositol (80 Ci/mmol, Amersham,
Denmark), extraction and separation of labeled inositol phosphates
after preincubation (5 min) with VIP or forskolin and subsequent
stimulation (5 min) with SP were performed as previously described
(19).
Statistical analysis
Results are expressed as means ± SEM unless
otherwise indicated. The data are representative of at least three
experiments performed under the same conditions. Student’s
t test, Mann Whitney U test, and one- or two-way ANOVA
followed by Bonferroni’s t test for multiple comparisons as
appropriate were used to compare differences between means.
P < 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, ß, and , but not
the and species (16). In female rats treated with
17ß-estradiol, PKC has been demonstrated as well by Western
blotting and by in situ hybridization (20). In
lactotroph-enriched cell cultures derived from male and female rats
grown in a steroid free environment used in the present study, the
species is not demonstrable, probably due to low levels of expression
of this isoform (17). The expression of PKC increases dramatically
after treatment with 17ß-estradiol of cell cultures derived from both
male and female animals for 24 h, but not the subtype
(authors’ unpublished observation). Accordingly, the presence of the
and species was not tested in the present study. IR in the
particulate fraction of PKC isozymes and ß from
lactotroph-enriched cell cultures is displayed in Fig. 1
. Challenge of cells with SP (100 nM) alone
resulted in selective translocation of the and ß subtypes (Fig. 1), whereas the and species were unaffected (Fig. 1). Addition
of VIP (100 nM) had no effect on the distribution of PKC
isozymes in resting cells, however, when cells subsequently were
exposed to SP they responded with a significant higher degree of
translocation than cells challenged with SP alone (Fig. 1). VIP alone
did not modulate the distribution of PKC of the and species
(Fig. 1).
|
). Pretreatment with
forskolin (10 µM) and subsequent stimulation of cells
with SP resulted in significantly increased redistribution compared to
cells exposed to SP alone (Fig. 1). When cells were pretreated with the
PKA inhibitor rp-cAMP (1 µM), the effect of VIP and
forskolin on SP-induced translocation of PKC and ß-IR was
abolished (Fig. 2). The potentiation displayed by VIP
and forskolin was totally dependent of preexposure of the compounds to
cells before addition of SP. Simultaneous challenge with SP and
VIP/forskolin did not result in potentiation of PKC isozyme
translocation (data not shown).
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and ß-IR and PKC
catalytic activity in the particulate fraction as a function of time is
given in Figs. 3 and 4. The rise in
PKC and ß-IR was followed by a concomitant rise in PKC catalytic
activity in this compartment. The IR as well as the catalytic activity
of PKC remained translocated for up to 10 min, whereafter levels fell
gradually to reach basal values at 20 min, except for cells pretreated
with VIP or forskolin where high levels of PKC and ß-IR, but not
the catalytic activity, were sustained throughout the observation
period. When tested on their own, VIP and forskolin did not affect the
PKC catalytic activity in the particulate fraction during an
observation period of 20 min (data not shown). However, a potentiating
effect at 2 and 5 min on the SP response was observed for both
compounds (Fig. 4).
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is displayed the influence of VIP and
forskolin on basal levels and on SP-induced formation of
[3H]Ins(1, 4, 5)P3 from the
precursor [3H]phosphatidylinositol 4,5-bisphosphate. Both
VIP and forskolin had no effect on production of
Ins(1, 4, 5)P3 on their own, whereas in cells
stimulated with SP (100 nM) and preincubated with either
VIP (100 nM) or forskolin (10 µM), the latter
two compounds significantly increased the
Ins(1, 4, 5)P3 response to SP. Inclusion of the
PKA inhibitor rp-cAMP blunted this potentiating effect.
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and ß-IR (16), did not
affect basal levels of cAMP in lactotroph-enriched cells. However,
preexposure of cells to SP and subsequent stimulation with VIP (both
100 nM) or forskolin (10 µM) caused a
significantly greater response than VIP or forskolin alone (Fig. 6, bottom, and Table 1).
Pretreatment of cells with TPA (100 nM) to down-regulate
PKC activity and subsequent exposure to SP and VIP still caused a minor
but significant increase in the level of cAMP compared with the effect
of VIP alone, but it was significantly less than the effect of SP in
VIP-stimulated cells not exposed to TPA treatment (Fig. 6, bottom). However, preexposure to the PKC-insensitive phorbol
ester 4--phorbol didecanoate (4--PDD) did not suppress the effect
of pretreatment with SP (Table 1). TPA, previously demonstrated to
translocate PKC, ß and in lactotrophs (16), increased basal
cAMP production significantly. When cells were stimulated
simultaneously by TPA and VIP (both 100 nM), the raise in
the concentration of cAMP was additive compared with effects of the
compounds alone (Fig. 6, top). The effect of TPA was
abolished by 24 h TPA, but not 4--PDD, pretreatment (Fig. 6, top, and Table 1).
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are shown the results of experiments where additional
manipulations of SP-potentiation of cAMP production by VIP or forskolin
were performed. Pretreatment of cells with the Ca2+
releasing compound thapsigargin (1 µM) had no effect on
the cAMP concentration. Combined with VIP (100 nM), the
response was not different from the effect of VIP alone, whereas the
SP-potentiation of the response to VIP was reduced but still
significantly different from the effect of VIP alone (Table 1). The
inhibitor of PKC staurosporine (1 µM) had the same effect
(reduced potentiation) as thapsigargin on the cAMP response to SP. In
cells preexposed to SP, dopamine (DA, 1 µM) was also able
to inhibit the potentiating effect of SP on VIP-induced cAMP
accumulation (Table 1). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The fact that the inhibitor of PKA, rp-cAMP, abolished the response of
VIP and forskolin on PKC isozyme translocation, renders PKA the most
probable effector substance. Because both the PKC and
Ins(1, 4, 5)P3 pathways were affected by
VIP/forskolin-stimulation, it is tempting to speculate that some common
regulatory point for these pathways is a target for PKA action, such as
the NK-1 receptor, the G protein, and/or PLC. PKA is known to
phosphorylate receptors linked to PLC like the
1-adrenoreceptor, especially when the receptor is
occupied by its ligand (23). However, phosphorylation of the receptor
by PKA would result in uncoupling of the receptor leading to a reduced
response (23, 32) and not the observed potentiation seen in the present
study leaving the NK-1 receptor a less likely candidate for PKA-induced
phosphorylation. Another possible target point is PLC, which is
expressed differentially in several isoforms in distinct cells and
tissues (24). Several investigators have reported that
PLC1 (but not PLCß 1 or 1)
is a target for PKA-induced phosphorylation, for example in C6Bu1 cells
(25), 3T3 cells (26) as well as the Jurkat T cell line (27). However,
in neither of these reports phosphorylation of PLC1 had
any effect on the catalytic activity of the enzyme and, furthermore,
the PLC1 is coupled to tyrosine kinase and not a
classical heterotrimeric G protein, making it difficult to extend these
observations to the results obtained in the present study. So far, no
data have been published demonstrating PKA mediated phosphorylation of
PLC species coupled to G proteins such as PLCß1 and
PLC. The G protein linked to the SP receptor, presumably belonging
to the Gq family (8), is a third candidate for PKA action because G
protein belonging to the Gi class Gi, has been demonstrated
to be phosphorylated by PKA, thereby altering the functional status of
this key point in signal transduction (28).
Other targets for PKA actions further downstream of the signal cascade includes the Ins(1, 4, 5)P3-3-kinase, which catalyses the formation of Ins(1, 3, 4, 5)P4 and alters its functional status when phosphorylated by PKA (29). The receptor for Ins(1, 4, 5)P3 contains also a phosphorylation site for PKA, thereby increasing the affinity of the receptor for its ligand resulting in increased Ca2+ release (30). However, further studies are needed to pinpoint specific phosphorylation sites in the PKC/Ins(1, 4, 5)P3 systems in lactotrophs. Another possibility is that PKA alters the activity of regulatory proteins not belonging to the PLC pathway. This hypothesis is favored by the observation that neither of the bifurcating limbs emerging from activated PLC were affected by VIP or forskolin when the NK-1 receptor was not stimulated. PKA-induced phosphorylation and thereby activation of calcium channels (31) is one possibility of a mechanism that may help explain this observation.
The translocation of PKC and ß by SP in cells preexposed to
VIP/forskolin was demonstrated immunologically. The results of this
experimental setting do not necessarily indicate that an increased
immunological redistribution of PKC isozymes parallels increased
translocation of PKC catalytically activity. However, in the time
course experiments there was a simultaneous translocation of both PKC
immunoreactivity and catalytic activity, indicating that redistributed
PKC and ß are catalytically active. Interestingly,
pretreatment of cells with VIP and forskolin induced a sustained
immunological translocation (after 10 min) of PKC and ß, which was
not reflected in the catalytic activity. This could represent a long
term regulatory mechanism by which adenylate cyclase coupled receptors
regulate PKC activity: rendering the isozyme plasma membrane-bound in
an inactive form instead of retranslocation to the cytosol where the
isozymes are accessible for repeated translocation to the plasma
membrane by SP-stimulation after a brief (5 min) period of
reconstitution (authors’ unpublished observations).
PKC activation and inhibition of the cAMP/PKA pathway have been studied
in a number of tissues and cells (for review see Refs. 32 and 33). PKC
has often been directly stimulated by application of phorbol esters
like TPA without focus on specific PKC isozyme involvement. However,
reports have been published where increments in cAMP by actions of
PKC-coupled agonists or TPA are associated with increased expression,
catalytic activity, and translocation of specific PKC isozymes (34, 35). We have previously demonstrated that SP and TPA translocate
PKC, ß and , ß and , respectively, in a well defined cell
system of enriched pituitary lactotrophs (16, 17). In the present
study, some of these results have been reproduced, but with concomitant
measurements of cAMP formation. Because TPA affected basal as well as
VIP/forskolin-induced cAMP formation, it is suggested that one or more
of PKC, ß and are involved. Even though TPA may have an effect
on cAMP by activating other signaling molecules than PKC, the
observations that the PKC inactive phorbol ester 4--PDD did not have
an effect on cAMP and that addition of the PKC inhibitor staurosporine
abolished the effect of TPA on cAMP, suggest that the indicated PKC
isozymes are involved in the cAMP response to TPA.
In contrast to TPA, SP did not affect cAMP when this pathway was not
activated by VIP or forskolin indicating a different mode of action by
SP as compared with TPA. One explanation for this could be that PKC
is responsible for the observed effect of TPA. On the other hand, SP
seems to use other signal pathways in affecting the cAMP concentration
because an effect of SP on cAMP was seen even after down-regulation of
PKC activity by TPA. As SP activates both limbs of the PLC pathway, we
employed thapsigargin as a tool for studying the
Ins(1, 4, 5)P3/Ca2+ route.
Thapsigargin, an inhibitor of the Ca2+-ATPase located in
the endoplasmatic reticulum, discharges
Ins(1, 4, 5)P3 sensitive Ca2+ stores
by turning off the reentry of Ca2+ (36). The results of the
thapsigargin experiments indicate that SP makes use of
Ins(1, 4, 5)P3 in the modulation of cAMP levels
because the potentiating effect of SP on VIP-induced cAMP accumulation
after 10 min of thapsigargin treatment was significantly reduced. This
suggests that Ca2+-dependent factors such as
Ca2+/calmodulin-dependent kinases, which are known to
modulate the functional status of the cAMP/PKA pathway (37), may be
involved further downstream. Interestingly, SP not only potentiated the
action of VIP on cAMP generation, but also the effect of forskolin,
which acts further downstream at the level of the catalytic subunit of
AC, indicating the existence of several target points for the action of
SP through PKC, ß and Ca2+. The receptor for VIP seems
not to be a target point for SP/TPA action because phosphorylation by
PKC species would uncouple the receptor leading to an inhibitory
response. The phosphodiesterase, which has been shown to be
phosphorylated by the action of TPA, thereby turning off the function
of this enzyme with a resulting rise in cAMP concentration (38), is
another possibility. The G proteins, Gs and Gi,
coupled to receptors that function through adenylate cyclase, are other
possible targets for PKC agonist-modulation of cAMP formation, which
have been demonstrated in a number cases (39, 40, 41). Because SP exerted a
stimulatory effect on the cAMP pathway, Gs is unlikely to
be a regulation point for SP action. However, Gi is a
possibility because phosphorylation at this site would abolish the
inhibitory input to adenylate cyclase. Therefore dopamine (DA) was
employed as a tool for studying possible involvement of Gi
as the D2 receptor localized on the lactotroph cell
membrane is coupled hereto. Still, the known inhibitory effect of DA on
VIP-induced cAMP accumulation was not altered by preincubation of cells
with SP, ruling out Gi as a target site. However, more
experiments are needed in the future to pinpoint regulation points and
mechanisms in the cross-talk between SP and VIP-coupled signaling
pathways.
Several reports have demonstrated that activation of PLC or PKC only modulates cAMP when receptors linked to adenylate cyclase are activated at the same time (42). Moreover, like in the present study, it has been shown that to regulate cAMP metabolism by PLC-coupled receptors, the PLC pathway has to be preactivated, emphasizing the need for the receptors to be occupied by their respective ligands to induce cross-talk (42). It is thus possible that activation of PLC by SP and the following stimulation of cAMP/PKA, which subsequently results in increased activity of the PLC signaling cascade, represents a long circuit stimulatory feed back mechanism in lactotrophs.
In summary, the present study has demonstrated that, in lactotrophs,
cross-talk exists between the Ins(1, 4, 5)P3/PKC
and cAMP/PKA messenger systems probed by SP and VIP, respectively. The
main effector substances in this cross-talk seem to be PKA, one or more
of PKC , ß, and Ca2+ released by
Ins(1, 4, 5)P3.
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Footnotes |
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Received September 9, 1996.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1- and ß2 adrenergic receptors by protein kinase C and
cyclic AMP-dependent protein kinase C. J Biol Chem 262:3106–3113 isozyme mediates both potentiation
and desensitization. J Neurochem 63:1361–1370[Medline]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
), forskolin (10 µM, 
) or vehicle
(
) and subsequently exposed to 100 nM substance P (SP,
t = 0) for a further period of 20 min. PKC catalytic activity was
determined by incorporation of 32P into a peptide
substrate, MBP4–14. Results are pooled from three
experimentse and are expressed as the mean ± SEM. *,
P < 0.05 vs. corresponding value
for vehicle (one-way ANOVA followed by Bonferroni’s t
test, n = 3).
