(Received for publication, September 21, 1995; and in revised form, December 28, 1995)
From the
Parafollicular (PF) cells secrete 5-hydroxytryptamine in
response to increased extracellular Ca (
[Ca
]
).
This stimulus causes Cl
channels in PF secretory
vesicles to open, leading to vesicle acidification. PF cells express a
plasmalemmal heptahelical receptor (CaR) that binds
Ca
, Gd
, and Ba
.
We now report that the CaR mediates vesicle acidification.
Ca
, Gd
, and Ba
induced vesicle acidification, which was independent of
channel-mediated Ca
entry. Agonist-induced vesicle
acidification was blocked by pertussis toxin, inhibitors of
phosphatidylinositol-phospholipase C, calmodulin, NO synthase, guanylyl
cyclase, or protein kinase G. PF cells contained NO synthase
immunoreactivity, and vesicles were acidified by NO donors and
dibutyryl cGMP. [Ca
]
,
and Gd
mobilized thapsigargin-sensitive internal
Ca
stores. [
S]G
and [
S]G
were
immunoprecipitated from PF membranes incubated with agonists in the
presence of [
S]adenosine
5`-O-(thiotriphosphate). Labeling of G
but
not G
was antagonized by pertussis toxin. Vesicles
acidified in response to activation of protein kinase C; however,
protein kinase C inhibition blocked calcium channel- but not
CaR-dependent acidification. We propose the following signal
transduction pathway: CaR
G
phosphatidylinositol-phospholipase C
inositol
1,4,5-trisphosphate
[Ca
]
Ca
/calmodulin
NO synthase
NO
guanylyl cyclase
cGMP
protein kinase G
opens
vesicular Cl
channel.
Two types of secretory vesicle are represented in synaptic
terminals. One is the small (50 nm) synaptic vesicle (SV) (
)that stores small molecule neurotransmitters(1) .
SVs can be replenished locally by endocytic recycling from the plasma
membrane (2, 3, 4) and thus are derived from
endosomes(5, 6) . The other type of vesicle, which is
also found in neuroendocrine cells, is a large, dense cored vesicle
that contains proteins and/or peptides packaged in the trans-Golgi
network and transported to terminals for regulated
secretion(7) . Paraneurons, such as thyroid parafollicular (PF)
cells, are endocrine cells that are closely related to
neurons(8) . They are derived embryologically from the neural
crest (9) and can be induced by nerve growth factor to assume a
neuronal phenotype in vitro(10) . The secretory
vesicles of PF cells share characteristics of both the large
trans-Golgi network-derived, peptide-containing dense cored vesicles
and the small, endosome-derived, low molecular weight
neurotransmitter-containing SVs. PF secretory vesicles resemble large,
dense cored vesicles in their size, initial formation in the
trans-Golgi network(11, 12) , and content of
calcitonin and other peptides(13) ; however, like SVs, PF
secretory vesicles contain a small molecule neurotransmitter,
5-HT(14, 15, 16) , and they
recycle(17) . In contrast to the peptides that are added to
vesicles at the time of their formation in the trans-Golgi network,
5-HT is loaded into PF vesicles by transmembrane transport from the
cytosol(15, 18) . As is generally true of monoamines
in SVs, this transport is mediated by a transporter protein in the
vesicular membrane (19) and driven by a transmembrane proton
gradient (18) that is established by the vesicular
H
-ATPase(20) . SV proteins, including the
synaptotagmin I, synaptophysin, and synaptobrevin(1) , are also
present in the membrane of PF secretory
vesicles(13, 48) .
PF secretory vesicles exhibit
properties that have not yet been detected either in neuronal SVs or
large, dense cored vesicles. The internal environment of PF secretory
vesicles is regulated by the same stimuli that initiate
secretion(18, 22, 48) . The interior of the
secretory vesicles of PF cells becomes acidic only when the cells are
stimulated by a secretogogue, such as increased extracellular
Ca (
[Ca
]
).
Because the membranes of PF vesicles are not permeable to
Cl
under resting conditions, influx of H
is limited by the generation of a transmembrane potential
gradient (
)(18, 22) . In response to
stimulation of PF cells with a secretogogue, a Cl
channel is opened in the vesicular membrane(18) . The
opening of this channel dissipates the
and permits
Cl
to enter vesicles as a counterion (48) so
that transport of H
is no longer electrogenic (18) and acidification of vesicles can proceed(22) .
The mechanism responsible for stimulus acidification coupling has
not yet been determined. A 64-kDa protein (p64), which is identical to
a Cl channel that has been cloned from epithelial
cells (23) , is present in the membranes of PF secretory
vesicles(48) . This protein has potential phosphorylation
sites, and stimulation of PF cells with a secretogogue increases the
phosphorylation of vesicular p64(48) . Acidification of
vesicles, moreover, is inhibited by compounds that antagonize a variety
of protein kinases or phosphatases (48) . These observations
are consistent with the following working hypotheses: (i) p64 is the
Cl
channel in vesicular membranes that permits the
secretion-induced entry of Cl
into vesicles; (ii) the
state and site of phosphorylation of p64 determine whether the channel
is open or closed; (iii) signal transduction pathways (still to be
identified) couple plasmalemmal secretogogue receptors to protein
kinases and/or phosphatases that determine the level of phosphorylation
of the channel protein. Because stimulus acidification and stimulus
secretion coupling can be dissociated(48) . The signal
transduction pathways responsible for each are not identical.
Because the acidification of secretory vesicles is evoked by
exposing PF cells to secretogogues, both acidification and secretion
could conceivably be mediated either by a cell surface Ca receptor or by a channel that is directly sensitive to the
[Ca
]
. Recently, each
of the [Ca
]
-sensitive
endocrine cells, parathyroid chief cells(24) , and PF
cells(49) , have been found to express a plasmalemmal calcium
receptor (CaR). The CaR is a heptahelical integral membrane protein
that when expressed in oocytes, couples to a pertussis toxin-sensitive
G protein(24) . Because
[Ca
]
evokes
secretion in PF cells and inhibits secretion in parathyroid cells, it
is possible that the CaR in each cell type is coupled to a different G
protein. The current experiments were undertaken to test the hypothesis
that the PF cell CaR is responsible for mediating the effects of
[Ca
]
on vesicle
acidification. Our data demonstrate that the acidification of the
secretory vesicles in PF cells is mediated via activation of the CaR
and that the CaR is coupled via G
and to a cascade of
second messengers, including inositol trisphosphate (IP
),
cytosolic free Ca
, NO, cGMP, and protein kinase G,
resulting in the phosphorylation and gating of the Cl
channel in the membranes of PF secretory vesicles.
In order to test the effect
of PTx on the binding of [S]GTP
S in
response to stimulation by ligand, membranous fraction obtained from PF
cells (50 µg in 100 µl) was preincubated with activated PTx (50
µg/ml) in the solubilization buffer containing NAD (100
µM), ATP (2 mM), EDTA (1 mM), and
dithiothreitol (1 mM) at 37 °C for 60 min. Membranes were
washed and incubated with 2.0 nM [
S]GTP
S. Immunoprecipitation of the
G
subunits were then investigated in control and
PTx-treated fractions.
Figure 1:
Acidification of PF vesicles in
response to CaR agonists is concentration-dependent. A, the
concentration effect relationship for the
[Ca]
-induced
acidification of vesicles. Note that the response does not saturate in
the absence of the calcium channel blocker nimodipine but does so in
its presence. B, the concentration effect relationships for
the acidification of vesicles induced by Gd
and
[Ca
]
+
nimodipine. Note that Gd
is a more potent agonist
than [Ca
]
and that the
effect of Gd
saturates. The percentage of cells
acidified in controls was 22.3 ± 4.5 % (n = 20).
The percentage of cells where the color was not clear was 5 ± 2
% (n = 20); these cells were not counted. Dead cells
were not fluorescent and appeared as
ghosts.
PF cell membranes were isolated and
stimulated with Ca or Gd
. Isolated
PF cell membranes were incubated with
[
S]GTP
S and exposed to Ca
(1 mM) or Gd
(250 µM).
The radioactivity immunoprecipitated by antibodies that react
specifically with G
, G
, or all known
G
subunits was determined. Baseline values were also
ascertained for control preparations, in which membranes were incubated
without agonists and in the presence of EGTA. The data were expressed
as the ratio of the radioactivity immunoprecipitated from membranes
incubated in the presence of agonists to that of the corresponding
controls in the same experiment. The effectiveness and specificity of
the antibodies were evaluated by immunoblotting (30) (Fig. 2). The antibodies we used for
immunoprecipitation (anti-G
i and anti-G
q) were specific in
that anti-G
i immunoprecipitated G
but not
G
, and anti-G
q immunoprecipitated G
but not G
. Antibodies to the membrane
Cl
channel, p64, failed to immunoprecipitate either
G
or G
. Both Ca
and
Gd
were found to stimulate the binding of
[
S]GTP
S to G
and, to a
much lesser extent, also to G
(Fig. 3A and B) (p < 0.01 for Gd
; p < 0.05 for Ca
versus control).
The concentrations of the agonists used in these experiments were each
supramaximal with respect to the acidification of vesicles (see Fig. 1B). At these concentrations, the degree of
stimulation of [
S]GTP
S binding to
G
and G
evoked by Ca
was not significantly different from that evoked by
Gd
. The sum of the radioactivity immunoprecipitated
by antibodies to G
and G
was equal to
that immunoprecipitated by antibodies that react with all known
G
subunits, suggesting that no additional G proteins
are stimulated by Ca
or Gd
. When
membranes were treated with PTx, the Ca
-induced
binding of [
S]GTP
S to G
was abolished (Fig. 3B); however, the stimulation
by Ca
of the binding of
[
S]GTP
S to G
was not
affected by PTx (Fig. 3B).
Figure 2:
Western blots showing the specificity of
antibodies used to immunoprecipitate G and
G
. Lane 1, immunoprecipitate obtained with
anti-G
q and probed with antibodies to G
; lane
2, immunoprecipitate obtained with anti-G
q and probed with
antibodies to G
; lane 3, immunoprecipitate
obtained with anti-G
i and probed with antibodies to
G
; lane 4, immunoprecipitate obtained with
anti-p64 (membrane Cl
channel) and probed with
antibodies to G
.
Figure 3:
Gd and
[Ca
]
activate
G
and G
. Membranes isolated from PF
cells were incubated with Gd
(250 µM) or
Ca
(1.0 mM) in the presence of
[
S]GTP
S. A, radioactive
G
and G
were immunoprecipitated from
membranes stimulated with Gd
. The data are expressed
as the experimental to control (no agonist) ratio, so that a value of
1.00 (dotted line) indicates no effect. Note that far more
[
S]G
than
[
S]G
was immunoprecipitated. B, [
S]G
and
[
S]G
were immunoprecipitated
from membranes stimulated with Ca
. Again, as with
membranes stimulated by Gd
,
[
S]G
[
S]G
. The activation of
G
but not that of G
was prevented by
preincubation of membranes with PTx. The experimental to control ratio
for G
stimulation by Ca
was 1.26, which
is a very small change but significantly different from 1.00 (t
= 2.479; p < 0.05). 1590 ± 250 cpm were
obtained in controls; following activation, the radioactivity
associated with the same amount of protein of G
was
6757 ± 1050 and 5962 ± 925 cpm for Gd
and Ca
, respectively. The radioactivity
associated with G
following activation was 2170
± 100 and 2105 ± 50 cpm for Gd
and
Ca
, respectively.
Figure 4:
Gd mobilizes
Ca
from an intracellular compartment in a
concentration-dependent manner. Cells were loaded with fura-2 and
exposed to Gd
in a Ca
-free medium.
The [Ca
]
was
determined by fluorescence. Note that there is good agreement between
the ED
for Gd
-induced acidification and
that for Gd
release of
[Ca
]
.
Figure 5:
[Ca]
can increase [Ca
]
independently of Ca
entry through
nimodipine-sensitive Ca
channels. Cells were loaded
with fura-2 and exposed to Ca
(10 µM-10
mM). The [Ca
]
was determined by fluorescence as in Fig. 3. The solid
line shows the [Ca
]
measured as a function of
[Ca
]
in the absence of
nimodipine, whereas the dashed line shows the
[Ca
]
as a function of
[Ca
]
in the presence
of nimodipine.
Figure 6:
Both the mobilization of
[Ca]
and the
acidification of secretory vesicles are inhibited by pretreatment of
cells with thapsigargin. A, the
[Ca
]
was measured in
fura-2-loaded cells ± thapsigargin pretreatment as a function of
time following challenge with Gd
. The rise in
[Ca
]
was prevented by
thapsigargin. B, the
[Ca
]
is shown as a
function of [Ca
]
.
Nimodipine was present to inhibit the entry of Ca
through L-type calcium channels.
[Ca
]
is lower than
control in thapsigargin-treated cells; however, at
[Ca
]
above 1
mM, a rise in [Ca
]
is seen despite the presence of nimodipine, possibly through
nonvoltage-gated cation channels. C, both
Gd
- and
[Ca
]
-induced
acidification of vesicles is antagonized by thapsigargin
(Gd
>
[Ca
]
).
Neither ryanodine (10
µM) nor caffeine (10 mM) increased
[Ca]
or acidified the vesicles
of PF cells (data not illustrated). These observations suggest that PF
cells lack ryanodine receptors and are consistent with the idea that
the CaR mobilizes Ca
from an IP
-sensitive
source. This hypothesis was tested. The CaR was stimulated with either
Gd
(250 µM) or a low concentration of
Ca
(1.0 mM), vesicle acidification was
measured, and the ability of U 73122, a specific inhibitor of
PI-phospholipase C, to antagonize the response was determined. U 73122
(10 µM) but not U 73343 (10 µM), an inactive
congener of U 73122, blocked the Gd
-induced
acidification of PF vesicles (Table 2). U 73122 also inhibited
the Gd
-induced increase in
[Ca
]
(Table 2). These
data confirm that PI-phospholipase C and thus IP
mediate
the increase in [Ca
]
that
follows stimulation of the CaR; moreover, the observations also provide
additional support for the idea that the rise in
[Ca
]
is a critical step in the
signal transduction pathway that leads to vesicle acidification.
Figure 7: NO synthase immunoreactivity can be detected in PF cells by immunocytochemistry. A, isolated PF cells show NO synthase immunoreactivity. B, PF cells show no immunostaining when antibodies to NO synthase are omitted and nonimmune serum is substituted. Bars, 15 µm.
Figure 8:
NO
appears to participate in signaling for Gd- and
[Ca
]
-induced vesicle
acidification. A, Gd
,
[Ca
]
, the NO donor,
sodium nitroprusside (1.0 mM), and dibutyryl cGMP (1.0
mM) all induce vesicle acidification. Imino-ornithine (10
µM), a NO synthase inhibitor, blocks the response to
Gd
and [Ca
]
but not that to sodium nitroprusside or dibutyryl cGMP. Shaded bars, no inhibitor; bars with dots,
imino-ornithine. B, LY 83583 (10 µM), a guanylyl
cyclase inhibitor, antagonizes vesicle acidification in response to
Gd
,
[Ca
]
, and sodium
nitroprusside but does not affect the response to dibutyryl cGMP.
Rp-cGMPS (10 µM), an inhibitor of protein kinase G,
antagonizes vesicle acidification induced by Gd
and
[Ca
]
. Lightly
shaded bars, no inhibitor; hatched bars, LY 83583; darkly shaded bars,
Rp-8-(4-chlorophenylthio)-cGMPS.
Figure 9:
PKC participates in mediating calcium
channel-dependent but not calcium channel-independent vesicle
acidification. A, the PKC activator, PMA (10 nM)
induces vesicle acidification equivalent to that induced by
Gd (250 µM). The response to PMA, but
not that to Gd
is blocked by the PKC inhibitor,
staurosporine, or by down-regulation of PKC. B, vesicle
acidification induced by a low concentration of
[Ca
]
(1.0
mM), which is channel-independent (see Fig. 1A), is unaffected by staurosporine (10
nM) or down-regulation of PKC (PMA, 10 nM;
overnight). In contrast, vesicle acidification induced by a high
concentration of [Ca
]
(5.0 mM), which is channel-dependent, is
antagonized by staurosporine or down-regulation of PKC. Note that the
degree of acidification induced by 5.0 mM [Ca
]
in the
presence of staurosporine or after PKC down-regulation is roughly
equivalent to that evoked by 1.0 mM [Ca
]
. Shaded
bars, no inhibitor; bars with thin hatching lines,
staurosporine; bars with thick hatching lines,
down-regulation.
Because studies with nimodipine
indicated that an increment in vesicle acidification above that induced
by the CaR occurred when Ca entered cells through
L-type calcium channels (see Fig. 1A), we tested the
possibility that PKC contributes to the Ca
channel-related acidification of vesicles. PF cells were exposed
to 5.0 mM [Ca
]
(rather
than 1.0 mM) in the absence of nimodipine, so that
Ca
would enter cells through calcium channels. When
this was done, vesicles acidified strongly (Fig. 9B).
Both staurosporine and down-regulation of PKC now significantly
antagonized vesicle acidification (Fig. 9B); however,
neither of these inhibitory treatments totally prevented acidification
of vesicles. Instead, the level of acidification induced by 5.0 mM Ca
in the presence of staurosporine or after PKC
down-regulation was reduced to a level that approximated that induced
by 1.0 mM Ca
(Fig. 9B).
These data are consistent with the idea that although PKC does not
mediate the basal acidification of vesicles induced by the CaR, it is
responsible for the Ca
channel-related increment in
vesicle acidification.
Figure 10:
Ba induces vesicle
acidification in nimodipine-pretreated PF cells. A, in the
absence of nimodipine, Ba
(2.5 mM) does not
induce a significant degree of vesicle acidification. In contrast, in
the presence of nimodipine, Ba
induces vesicle
acidification equivalent to that evoked by Ca
(1.0
mM; ± nimodipine) or Gd
(250
µM). Black bars, nimodipine absent; hatched
bars, nimodipine present. B, 2.5 mM Ba
strongly inhibits acidification in response
to 5.0 mM Ca
but not that in response to 250
µM Gd
. In fact, vesicle acidification in
response to Gd
and Ba
is greater
than that evoked by either ion alone, suggesting that the effects of
Gd
and Ba
on vesicle acidification
are additive. Black bars, Ba
absent; hatched bars, Ba
present.
The current study was undertaken to determine whether the CaR
is responsible for the phenomenon of secretogogue-evoked acidification
of secretory vesicles in PF cells and if so, to characterize the signal
transduction pathway. It was, however, first necessary to distinguish
effects mediated by the CaR from those resulting from the entry of
Ca through plasmalemmal calcium channels.
Gd
was particularly valuable for this purpose.
Gd
is both an efficient ligand at the CaR (8, 24) and a highly effective calcium channel
blocker(39) ; therefore, when Gd
is used to
activate the CaR, responses are not complicated by the influx of
Ca
through calcium channels. Gd
was
found to be an agonist that induced vesicle acidification in a
concentration-dependent manner. A similar, but more complicated
response was elicited by [Ca
]
.
At low concentrations of [Ca
]
(
1.0 mM), vesicle acidification was unaffected by
the addition of calcium channel blockers and the concentration effect
curve was roughly parallel to that of Gd
, although it
was shifted to the right. These data suggest that vesicle acidification
in response to low concentrations of
[Ca
]
and Gd
is the result of an action at a common receptor and that
Gd
is a more potent agonist. At higher concentrations
of [Ca
]
, however, an increment
in acidification occurred that was beyond that induced by
Gd
. This increment was blocked by the L-type calcium
channel blocker, nimodipine. It is possible that this increment
represents the influx of Ca
in a subpopulation of
cells that does not respond to low concentrations of
Ca
. These observations suggest that acidification of
PF vesicles can be induced by mechanisms that are both independent of
and dependent on plasma membrane calcium channels.
A third CaR
agonist, Ba, provided further insight into the nature
of calcium channel-independent vesicle acidification. By itself,
Ba
causes PF cells to secrete, but it does not induce
vesicle acidification(48) . This observation provided the
initial evidence that acidification of vesicles is not a requirement
for secretion by PF cells and that the transduction mechanisms
responsible for vesicle acidification and secretion are not identical.
When added together with nimodipine, however, Ba
caused vesicles to acidify; moreover, the response to
Ba
was synergistic with that of Gd
.
In contrast, in the absence of nimodipine, Ba
inhibited the vesicle acidification induced by low concentrations
of [Ca
]
. These observations
suggest that Ba
, like Gd
and
[Ca
]
, can cause vesicles to
acidify through a mechanism that is independent of calcium channels.
This effect of Ba
, however, is only manifested when
Ba
is prevented from entering PF cells. If
Ba
enters cells, as it does in the absence of
calcium, channel blockade can evidently inhibit signal transduction
from a plasmalemmal receptor. This intracellular action accounts for
the ability of Ba
to inhibit the action of
[Ca
]
when Ba
is allowed to enter PF cells. In the absence of nimodipine,
therefore, the plasmalemmal action of Ba
is probably
masked by a counteracting intracellular effect. The intracellular
inhibitory action of Ba
, however, is not manifest
when Ba
is applied together with
Gd
, which prevents the entry of Ba
through calcium channels. The observation that each of three CaR
agonists, Ca
, Gd
, and
Ba
can induce vesicle acidification by a plasmalemmal
action that is independent of calcium channels, supports the idea that
the CaR is responsible for their common effect. The
nimodipine-dependent increment in vesicle acidification induced by
concentrations of [Ca
]
above
1.0 mM suggests that Ca
entry through L-type
calcium channels can also contribute to vesicle acidification through a
process that does not involve the CaR.
If the CaR is responsible for
agonist-induced vesicle acidification, then stimulus-acidification
coupling would be expected to be mediated by a signal transduction
pathway that involves a G protein(8, 24) . The
identification of such a pathway would thus simultaneously provide
evidence for a role of the CaR in vesicle acidification and also
provide important insight into the responsible mechanism. We thus
tested the roles played by G proteins, likely second messengers, and
effectors in mediating vesicle acidification in response to CaR
agonists (Gd or a low concentration of
[Ca
]
in the presence of
nimodipine). Vesicle acidification induced by Gd
and
Ca
was abolished by PTx, and these agonists also
activated G
(as determined by measuring the binding of
[
S]GTP
S to immunoprecipitated
G
). Although the agonists were also found to activate
G
, this effect was not, like that of the activation of
G
, inhibited by PTx. The ability of Ca
to
activate Gq was slight but significant (p < 0.05 when each
sample was compared with its own control). It thus seems likely that
coupling of the CaR to G
is a critical step in stimulus
acidification coupling. The coupling of the CaR to G
in PF
cells is similar to the coupling of the receptor expressed in
oocytes(24) . Coupling of the CaR to G
may be
involved in other responses of PF cells to stimulation of the receptor.
Stimulus-induced acidification, for example, is distinct from
stimulus-induced secretion, which is resistant to PTx(48) .
Several observations supported the idea that the G protein activated
by the CaR stimulates PI-phospholipase C. Both Gd and
low concentrations of [Ca
]
mobilized Ca
from an internal pool that was
sensitive to thapsigargin. Thapsigargin inhibits the calcium pumps of
the ER, preventing the reuptake of Ca
, which leads to
an irreversible depletion of IP
-mobilizable internal
Ca
stores(32) . Because ryanodine did not
mobilize Ca
, PF cells probably lack ryanodine
receptors; therefore, cyclic ADP-ribose, which acts through these
receptors(40) , is not likely to be involved in the
CaR-stimulated mobilization of Ca
from internal
stores. More direct evidence for the participation of PI-phospholipase
C was obtained with the PI-phospholipase C inhibitor, U
73122(4) , which specifically blocked both the mobilization of
[Ca
]
and the acidification of
vesicles in response to Gd
or
[Ca
]
. These data suggest that
PI-phospholipase C activated secondary to the coupling of the CaR to
G
causes the release of IP
, which mobilizes
[Ca
]
. Although the ability of
the
subunits of the G
family of G proteins to
activate PI-phospholipase C has been demonstrated directly(3) ,
it has been difficult to demonstrate PI-phospholipase C activation by
the
subunits of PTx-sensitive G
proteins(5, 41, 42) ; nevertheless,
receptor-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate
is blocked by PTx in many different types of
cell(2, 41, 43) . Many reports have indicated
that the
subunits, rather than the
subunits, are the
components of PTx-sensitive G proteins, including G
, which
activate PI-phospholipase C(41, 42, 44) . It
is thus possible that the
subunits released from G
following its coupling to the CaR are responsible for the
CaR-dependent activation of PI-phospholipase C. A role for
G
, however, cannot be ruled out. Both the
and
the
subunits of G
have been found to participate
in the adenosine A1 receptor-mediated activation of PI-phospholipase
C(45) .
Thapsigargin not only antagonized the
agonist-induced increase in
[Ca]
, but also antagonized
stimulus acidification coupling, suggesting that the PI-phospholipase
C-mediated increase in [Ca
]
is
a critical component of the transduction pathway. It seems likely that
the effect of the mobilization of
[Ca
]
is mediated by activation
of NO synthase. PF cells were found by immunocytochemistry to contain
NO synthase (and NADPH diaphorase activity), which is known to be
activated by Ca
/calmodulin(46) ; moreover,
the NO synthase inhibitor, imino-ornithine, blocked the vesicle
acidification induced by Gd
or Ca
but not that induced by the NO generator, sodium nitroprusside.
In addition, as expected for an effect mediated by NO
synthase(28) , Gd
- and
Ca
-induced vesicle acidification were antagonized by
an inhibitor of guanylyl cyclase (LY 83583, which also blocked the
effects of sodium nitroprusside) and G kinase (Rp-cGMPS). The
involvement of NO synthase in stimulus acidification could account for
the previous observation that inhibition of calcineurin, a
Ca
/calmodulin-dependent protein phosphatase,
antagonizes Ca
-stimulated vesicle
acidification(48) . Phosphorylated NO synthase is a substrate
for calcineurin, and phosphorylation of NO synthase decreases its
catalytic activity(47) . The proposed signal transduction
pathway for CaR-induced vesicle acidification is shown in Fig. 11.
Figure 11:
A model showing the proposed signal
transduction pathway leading to the acidification of parafollicular
vesicles in response to extracellular Ca or
Gd
.
Although the evidence outlined above supports the
hypothesis that the CaR is coupled to vesicle acidification,
stimulation of this receptor is not the only means by which vesicle
acidification is controlled. It is probable that the entry of
Ca through nimodipine-sensitive L-type calcium
channels also causes vesicles to acidify, that this effect is
independent of the CaR, and that the Ca
channel-related and CaR-induced components of vesicle
acidification are additive. Thus, a nimodipine-inhibitable increment in
acidification is seen when Ca
is present at
concentrations above that needed to stimulate the CaR. The magnitude of
the CaR-mediated component of acidification can be estimated either
from the maximal response to Gd
or from that to
Ca
in the presence of nimodipine, which is about the
same. The observation that the calcium channel-related component of
vesicle acidification, but not that which appears to be stimulated by
the CaR, is blocked by inhibitors of PKC (staurosporine and calphostin
C) and by PKC down-regulation confirms that independent mechanisms are
responsible for the two components. The diacyl glycerol that is
generated by the action of PI-phospholipase C activated in response to
CaR stimulation could activate PKC. Diacyl glycerol activation,
however, seems to be insufficient for vesicle acidification. The data
are consistent with the idea that the component of vesicle
acidification related to calcium channels is mediated by PKC, whereas
that related to the CaR is mediated by a G kinase. In both cases,
phosphorylation of p64, the chloride channel in the membranes of PF
vesicles could be responsible for increasing the Cl
conductance that underlies acidification(48) .
The
role played by the CaR in normal homeostasis remains to be determined.
[Ca]
was observed to activate
the receptor in concentrations that are below those normally found in
extracellular fluid. The effect of such low concentrations of
[Ca
]
, however, were only
detected in comparison to cells incubated in media containing EGTA,
which are almost Ca
-free. It is possible that the CaR
is partially desensitized under physiological conditions and thus
responds in vivo only to higher than normal concentrations of
[Ca
]
. If so, it is likely that
secretogogue-induced vesicle acidification will be mediated both by the
CaR and the entry of Ca
through L-type calcium
channels, which was found to occur at concentrations of >1.0
mM. PF cells are depolarized by exposure to greater than
resting concentrations of
[Ca
]
(21) . It is
conceivable that stimulation of the CaR contributes to the
[Ca
]
-induced depolarization of
PF cells. Whether or not this is so, the depolarization is probably
linked to the opening of L-type calcium channels, which are
voltage-dependent, and thus to the activation of the parallel
PKC-dependent pathway of vesicle acidification.
The function of the
phenomenon of stimulus-induced vesicle acidification also needs to be
clarified. The uptake of 5-HT from the cytosol, where it is
synthesized, into the vesicles in which it is stored is driven more by
the proton gradient than by the potential difference across the
vesicular membrane(18) . Most of the vesicles of nonstimulated
PF cells are not acidic because the development of a membrane potential
difference limits acidification until the vesicular Cl channel opens in response to stimulation. It thus seems likely
that the loading of 5-HT into vesicles is episodic and greatest after
stimulus-induced vesicle acidification. The potential-driven uptake of
5-HT into the vesicles of resting cells is probably small in comparison
with the pH gradient-driven uptake into the vesicles of
secretogogue-stimulated cells. In fact, this enhancement of 5-HT uptake
by secretogogue stimulation has actually been
demonstrated(18) . In contrast, the stability of 5-HT within
vesicles may be decreased and its osmotic activity may be increased by
acidification. PF vesicles contain a matrix protein, serotonin-binding
protein, that binds 5-HT in a neutral but not acidic
medium(16, 22) . Acidification would thus be expected
to facilitate the secretion of 5-HT. Serotonin-binding protein is
retained by secretory vesicles, even though they recycle(17) .
Serotonin-binding protein, therefore, probably remains membrane-bound
during exocytosis and is recaptured with the vesicular membrane at
endocytosis. Although exposure to secretogogues does not cause the
fusion of all secretory vesicles with the plasma membrane, it does
cause virtually all vesicles to become acidic(22) . Vesicles
can thus load episodically with 5-HT, which is retained during the
intersecretory period. Retention may be facilitated by binding to
serotonin-binding protein or other matrix components. Vesicle
acidification may also contribute to the processing of calcitonin,
somatostatin, or other products within vesicles(25) .