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(Received for publication, June 7,
1995; and in revised form, January 2, 1996) From the
Expression of human parathyroid hormone receptor (hPTHR) was
obtained in Xenopus oocytes. Receptor function was detected by
hormone stimulation of endogenous Ca Parathyroid hormone (PTH) ( The transfection
experiments demonstrated that the PTHR can activate multiple signaling
events in the same cell, including production of cAMP, activation of
phospholipase C, and elevation of cytosolic
Ca The signaling systems
responsible for elevation of cytosolic Ca These
studies indicate a need for further examination of signaling by the
PTHR under conditions where different signals can be studied
independently in the same cell system. The functional expression of the
PTHR in Xenopus oocytes appears to offer that possibility.
Specifically, the frog oocyte has a Ca
Intracellular injection of InsP
Figure 1:
Functional expression of human
parathyroid hormone receptor in Xenopus oocytes. Whole cell
voltage clamp analysis of oocytes microinjected with cDNA or mRNA of
the human parathyroid hormone receptor. A, membrane current
traces recorded at a holding potential of -80 mV. Inward current
is downward. 3 µl of 2.43
Figure 2:
Human
parathyroid hormone receptor-mediated current is dependent on
intracellular Ca
Figure 3:
Human
parathyroid hormone receptor induces a Cl
Figure 4:
Human
parathyroid hormone receptor-induced current is not mediated by cAMP.
Oocytes expressing the hPTHR were voltage-clamped at -80 mV.
Microinjection of cAMP or its analogues 8-Br-cAMP, 8-CPT-cAMP, and
2,3-cAMP did not induce any inward current. hPTH was subsequently
applied to confirm the oocytes did have hPTH-induceable
response.
Figure 5:
Human parathyroid hormone
receptor-mediated current has a long latency of onset and a slow
kinetics. A, diagram showing the measurement of the latency
time and the start-peak time. Top trace shows an hPTH-induced
response. As a comparison, the bottom trace shows a serotonin
(5-HT)-induced membrane current in an oocyte injected with the RNA for
5-HT
On average, the chloride current
responses to serotonin are also larger than those to PTH. However, this
quantitative difference cannot be interpreted since it could be due to
one or a combination of several variables including the efficiency of
expression of the receptor message, the stability of the injected
nucleic acid messages, the stability of the expressed receptor in the
cell membrane, the efficiency of coupling to second messenger systems,
and the sensitivity and size of responsive calcium compartments. The
relative levels of receptor expression were examined in ligand binding
studies. We found that the levels of expression of hPTH receptor and
5-HT
Figure 6:
hPTH and 5-HT responses do not
cross-desensitize each other. Oocytes were voltage clamped at -80
mV. A, membrane current traces of first and second 5-HT (upper trace) or hPTH (lower trace) stimulation,
showing that activation of each receptor induces complete homologous
desensitization. B, membrane current trace induced by 5-HT and
hPTH in a single oocyte coinjected with 5-HT
Figure 7:
Xenopus oocytes expressing the
PTHR can be desensitized to injected InsP
Since
the PTHR can activate Ca
Figure 8:
Injected NADP
As with other hormone receptor systems that have been
expressed in
oocytes(24, 25, 26, 27) , this work
shows that increases in cytosolic Ca Earlier
studies indicated that PTH can activate increases in cytosolic
Ca This work is the first demonstration of the
cytosolic Ca The most
interesting results from this work are the apparent lack of involvement
of InsP An alternative
interpretation is that frog oocytes possess Ca Although InsP The inhibition
of PTHR signaling by NADP
Volume 271,
Number 14,
Issue of April 5, 1996 pp. 8183-8191
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
EVIDENCE FOR A NOVEL SIGNALING PATHWAY (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-activated
Cl
current. This current was blocked by injected, but
not by extracellular, EGTA, confirming that the hPTHR activates
cytosolic Ca signaling pathways. PTH responses were
acutely desensitized but were regained in 6-12 h. Injection of
cAMP or analogues had no effect on either responsiveness or
desensitization to hPTH. The hPTH response was more sluggish than seen
with serotonin 5-hydroxytryptamine (5-HT
) receptor. In
oocytes co-expressing both hPTHR and 5-HT receptors,
homologous desensitization was seen, but cross-desensitization was not
observed. Injection of inositol 1,4,5-trisphosphate (InsP
)
elicited a fast inward current similar to that induced by serotonin,
and complete cross-desensitization occurred between the InsP and 5-HT responses. Desensitization by hPTH did not
affect responses to either InsP or serotonin, but cells
desensitized to injected InsP still responded strongly to
PTH. Oocytes did not respond to either cADPR or NAADP
,
but NADP and analogues were found to be potent
inhibitors of PTH signaling. We suggest that PTH cytosolic
Ca signaling in oocytes either involves a novel
signaling system or proceeds through a Ca compartment
whose responsiveness is regulated in a novel way.
)is the primary regulator
of calcium and phosphate homeostasis in higher animals(1) .
This 84-amino acid peptide and truncated forms such as the 1-34
or 1-36 fragments initiate the biological actions of PTH through
a specific receptor (PTHR) on the plasma membrane in target tissues,
primarily kidney and
bone(2, 3, 4, 5) . Recently, the
cDNAs of PTHR from rat, mouse, and human cells have been
cloned(6, 7, 8, 9, 10) ,
and both transient and stable transfection of PTHR has been
described(7, 11, 12) .(7, 12) . However, this multiple
signaling capability is not always present in cells that
constituitively express the PTHR. In some systems, a cAMP response is
observed in the absence of the cytosolic Ca response(13, 14) , while in others the reverse
appears to be true(15) . In addition, N-terminally truncated
forms of PTH that do not activate adenylyl cyclase still appear to
generate the cytosolic Ca signal(16) , and
likewise cells in a cultured bone cell line desensitized to PTH with
regard to the cAMP response still show a cytosolic Ca signal(17) . Thus, the factors that determine signaling
in different systems are not understood. in response
to PTH are also complex. The hormone has been reported to activate
Ca influx through a cAMP sensitive plasma membrane
channel(18) , but elevated cytosolic Ca can
also be observed in the absence of influx of extracellular
Ca(19) . The latter effects have been
attributed to generation of InsP through the action of
phospholipase C(20, 21) . However, some studies
suggest that production of InsP is not obligatory for
elevation of cytosolic Ca by PTH. It has been
reported that parathyroid hormone and thrombin release intracellular
calcium from different calcium stores in osteoblast-like UMR 106-H5 rat
osteosarcoma cells(22) , and no effect of PTH on InsP was found in this study. It has also been reported that
full-length hPTHR stimulated by hPTH does not increase InsP levels in stably transfected HK-293 cells, while some truncated
forms of the receptor do express this activity (23) .-activated
Cl channel that is directly utilized to detect
cytosolic Ca changes (24, 25, 26, 27) but no endogenous
cAMP-activated channels. Also, the ability for direct manipulation of
cytosolic contents provide a flexible system that may be used to
clarify the PTH signaling system. In this paper, we demonstrate its use
for examination of cytosolic Ca signaling.
Materials
Human parathyroid hormone receptor
cDNA in pcDNA I vector was generously provided by Dr. G. V. Segre
(Massachusetts General Hospital/Harvard Medical School, Boston). D-myo-Inositol 1,4,5-trisphosphate, 8-Br-cAMP,
8-CPT-cAMP, c-2,3-AMP, and serotonin (5-hydroxytryptamine (5-HT)) were
purchased from Sigma; hPTH (1-34) was from Bachem, and L-15
medium was from Life Technologies, Inc.Cloning of Human Parathyroid Hormone Receptor into
Expression Vector and in Vitro RNA Synthesis
cDNA of human
parathyroid hormone receptor was subcloned into pcDNA3 expression
vector (Invitrogen) at BamHI-XbaI sites for DNA
injection to the oocytes. For in vitro synthesis of RNA, the
subclone was linearized by XbaI, and cRNA was synthesized with
T7 mMessage mMachine kit (Ambion). The RNA yield was measured by A
and 1-10 ng of RNA was injected into
each oocyte.Preparation of Xenopus Oocytes
Adult female Xenopus laevis frogs were purchased from commercial sources (Xenopus I, Ann Arbor, MI, and African Fish Farm, South
Africa). Frog was anesthetized in 0.17% MS-222 (tricaine
methanesulfonate) for about 1 h. Ovarian tissue was surgically removed,
and the follicular cells were removed by treatment with 2 mg/ml of
collagenase in Ca-free ND96: 96 mM NaCl, 2
mM KCl, 1.5 mM MgCl
, and 5 mM HEPES, pH 7.5. Stage V and VI oocytes were selected and kept in
50% L-15 medium supplemented with 0.8 mM glutamine and 10
µg/ml gentamicin at 18-20 °C.Microinjection of DNA and RNA
Injection of oocytes
was performed in a Ca-free ND96 solution by an
automatic Drummond microinjector. 5-10 nl of DNA or 30-40
nl of RNA solution per oocyte were injected. After injection, oocytes
were transferred to 50% of L-15 medium for incubation.Whole Cell Voltage Clamp
Electrophysiological
recording was essentially as described
previously(29, 30) . Briefly, oocytes placed in a
recording chamber were voltage-clamped at -80 mV with two
electrodes (filled with 3 M potassium chloride and having a
resistance of 0.5-3 megaohms) using an Axoclamp-2A (Axon
Instruments). Oocytes were superfused with ND96 containing 1.8 mM of CaCl. hPTH (1-34) was directly applied to the
bath, and 100 nM of 5-HT was applied to the oocytes by
superfusion. Voltage command protocols were generated by pCLAMP
software (Axon Instruments), and the resulting membrane current changes
were recorded using the same program and on a chart recorder. , EGTA, cAMP, 8-Br-cAMP,
8-CPT-cAMP, and 2`,3`-cAMP were performed using an automatic Drummond
microinjector, similar to that described by Lin et
al.(31) . InsP (4.8 mM), EGTA (50
mM), cAMP (10 mM), 8-Br-cAMP (10 mM),
8-CPT-cAMP(10 mM), and 2`,3`-cAMP(10 mM) were
dissolved in distilled water and back filled into the pipette (tip
broken to about 5 µm diameter) of the microinjector. A few droplets
of the solution were expelled out of the pipette just before the oocyte
impalement to ensure a free flow of the injection pipette. A volume of
10 nl for InsP, EGTA, cAMP, 8-Br-cAMP, 8-CPT-cAMP, or
2`,3`-cAMP was injected into individual oocytes. Oocytes were perfused
with calcium-free NP96 solution during all injections. After recording,
injection pipettes were examined under the microscope to ensure that
the pipettes were not clogged. For InsP injection, shortly
after injection into experimental oocytes, InsP was also
injected into control oocytes to ensure that InsP was still
capable of eliciting a response.Receptor Binding Studies
Radioligand binding to
expressed receptors on oocytes was performed essentially as described
by Yoshii et al.(28) . Oocytes were prewashed once in
ND96 solution and transferred to 1 ml of ND96 containing 5 nM of either [H]mesulergine or 
I-hPTH (1-34 tyrosine amide) for 90-min incubation
at room temperature. Mianserin (5 µM) or hPTH (1-34)
(10 µM) were used as competing ligands to define the
amount of specific binding for 5-HT receptor or hPTH
receptor, respectively. Oocytes were pooled in groups of 5 or 10 for
the binding studies, which were done in duplicate. Binding was
terminated by vacuum filtration through Whatman GF/B filters pretreated
with 1% polyethylenimine. The filters were washed 3 times with 3.5 ml
of ice-cold 50 mM Tris-HCl, pH 7.4, and then counted.
Functional Expression of Human Parathyroid Hormone
Receptor in X. laevis Oocytes
To express the hPTHR in Xenopus oocytes, a DNA clone for the hPTHR (10) was
used. Aliquots of cDNA of the hPTHR were microinjected into the nucleus
of defolliculated X. laevis oocytes. Alternatively, synthetic
cRNA was in vitro transcribed from the cDNA clone and
microinjected into the cytoplasm of Xenopus oocytes. 3-4
days after cRNA or cDNA injection, stimulation with 1-34 hPTH
elicited an inward current in hPTHR-injected oocytes but not in
uninjected control cells (Fig. 1A). The hPTH response
displays a strong homologous desensitization, as shown by a lack of
response from the second hPTH stimulation following washing out the
bath hPTH used in the initial stimulation (Fig. 1A, middle trace, and Fig. 1B). The responsiveness
of hPTHR-expressing oocytes slowly recovers, with the size of the
inward current showing a partial recovery after 6 h (Fig. 1A, bottom trace, and Fig. 1B) and a complete recovery after 12 h (data not
shown). The hPTHR was consistently expressed in oocytes, whether DNA or
RNA was injected.
10
M hPTH (1-34) was directly added to the recording chamber with
a capacity of about 500 µl so that the final concentration of hPTH
in the recording chamber was
10
-10
M. Applying
hPTH to uninjected oocytes, no response was observed (top
trace). When hPTHR-injected oocytes were stimulated with hPTH,
inward currents were induced (middle and bottom
trace). After the first hPTH stimulation, the oocyte was washed
with ND96 with 1.8 mM CaCl. When a second hPTH was
applied about 1 min after the washout, no response was elicited (middle trace). When the second hPTH stimulation was applied
several hours after the washout, the response was partially recovered (bottom trace). B, bar graph showing hPTHR-mediated
membrane currents and homologous desensitization. hPTH did not induce
noticeable membrane currents in uninjected oocytes (control).
Following the first hPTH stimulation, a large inward current was
observed (581.3 ± 170 nA, n = 4). * indicates a
significant difference from the control value (p < 0.01).
Upon washout of hPTH, very little response could be induced by a second
application of hPTH within 5 min, indicating complete homologous
desensitization. The response slowly recovered after several hours
(257.5 ± 85.7 nA, n = 4).** indicates a
significant difference from the 5-min washout value (p <
0.01).
Impact of Calcium for the Human Parathyroid Hormone
Receptor-mediated Current
To determine whether the hPTH-induced
current in oocytes requires cytosolic Ca, 500 pmol of
Ca chelator EGTA was microinjected into hPTHR
expressing oocytes. This gives a final concentration of EGTA at
0.5 mM for oocytes with an average volume of 1 µl.
Stimulation with hPTH (1-34) after EGTA injection produced no
responses (Fig. 2A, middle trace, and Fig. 2B), indicating that the hPTH-induced currents
were completely eliminated by intracellular EGTA injection. However,
the addition of 1 mM EGTA to the calcium-free ND96 superfusion
solution had no effect on hPTH-induced currents (Fig. 2A, bottom trace, and Fig. 2B). This showed that the hPTHR-induced response
in Xenopus oocytes is dependent on intracellular
Ca but not on extracellular Ca.
and independent of extracellular
Ca. Oocytes expressing hPTHR were voltage clamped at
-80 mV, and hPTH application was as described in the legend to Fig. 1. A, in normal Ringer's solution (ND96
containing 1.8 mM Ca), hPTH induces an
inward current (top trace). When 10 nl of 50 mM EGTA
was injected into the cell just before the application of hPTH,
hPTH-induced response was abolished (middle trace), indicating
a dependence of the hPTHR response on intracellular
Ca. When the extracellular Ca was
removed by bathing oocytes in Ca-free ND96 containing
1 mM EGTA, hPTH application resulted in an inward current (bottom trace) that is similar to hPTH response in normal
[Ca]
(see top
trace), indicating that hPTH-induced current is independent of
extracellular Ca. B, bar graph summarizing
the effect of Ca on hPTH-induced current. 1.8
mM [Ca
]
:
hPTH-induced currents in hPTHR-expressing oocytes in normal
extracellular Ca. The amplitude of the current is
473.4 ± 102.6 nA (mean ± S.E., n = 8). EGTA injection + 1.8 mM [Ca]:
response from oocytes bathed in normal extracellular Ca and microinjected with EGTA before hPTH stimulation. * indicates
a significant difference from the non-EGTA injected group (p<0.01). 0 mM [Ca]:
current response from oocytes bathed in Ca-free
solution containing 1 mM EGTA. The response amplitude is 453.3
± 98.7 nA (mean ± S.E., n =
3).
The Inward Current Is Mainly Carried by the Chloride
Ion
The ionic selectivity of hPTH-induced currents were
determined using different channel blockers. There was no effect on
hPTH-induced membrane currents when in the bath solution Ca was chelated with EGTA and, with the inclusion of Cd as the Ca channel blocker and 4-AP and TEA as
the K channel blocker (Fig. 3A, upper trace, and Fig. 3B). However, the
membrane current could be completely suppressed by using an external
ND96 solution containing 1 mM niflumic acid, a Cl channel blocker (Fig. 3A, lower trace,
and Fig. 3B). It has been reported that reversal
potential for Cl channel is approximately -25
mV(35) . The hPTH-induced membrane current was also eliminated
by clamping the oocytes at the holding potential of -25 mV (data
not shown). Together, these results indicate that the hPTHR-induced
current is mainly carried by the Cl ion.
current in
oocytes. The membrane current traces were recorded at a holding
potential of -80 mV in oocytes expressing hPTHR. A,
representative traces from oocytes bathed in modified Ringer's
solution containing EGTA, Cd, 4-AP, and TEA (upper trace) of niflumic acid (lower trace). B, comparison of the hPTH induced responses in the presence of
different channel blockers. ND96, control group recorded in
normal ND96. The amplitude is 675 ± 49 nA (mean ± S.E., n = 5). EGTA, Cd, 4-AP, TEA,
response recorded in a modified Ringer's solution containing
Ca channel blocker Cd (100
µM) and K channel blocker 4-AP (5
mM) and TEA (36 mM). 1 mM EGTA was used to
chelate extracellular Ca to prevent Ca influx-induced currents. The amplitude is 670 ± 44 nA
(mean ± S.E., n = 5). Niflumic acid,
response recorded in ND96 containing the Cl channel
blocker niflumic acid (0.4 mM). The amplitude is 28 ±
14 nA (mean ± S.E., n = 5). * indicates a
significant difference from the control group (p <
0.01).
Human Parathyroid Hormone-induced Response Is Independent
of cAMP
It has previously been reported that PTH can activate
production of cAMP in Xenopus oocytes injected with mRNA
extracts from target cells that contain the PTHR message(32) .
Also, in some systems, cAMP has been reported to mediate
desenstitization to PTH by a protein kinase A-dependent
process(33, 34) . Therefore it was of interest to
examine whether cAMP has any effect on the cytosolic Ca response to PTH in our system. Intracellular injection of cAMP
and cAMP analogues, 8-Br-cAMP, 8-CPT-cAMP, and 2`,3`-cAMP, did not
induce any membrane current in either control oocytes not expressing
the hPTHR (data not shown) or hPTHR-expressing cells (Fig. 4).
These same hPTHR-expressing cells can produce an inward current by
subsequent hPTH application, demonstrating that they are not
desensitized to PTH by cAMP, and suggesting that the desensitization
process in oocytes is not mediated by protein kinase A.
The Response to PTH Is More Sluggish Than That of a
Receptor Coupled to the Phospholipase C Pathway
Xenopus oocytes have been used widely to study membrane receptors that
couple to the phospholipase C signaling pathway(24) , since
stimulation of such a receptor expressed in oocytes gives a robust
inward Cl current. To compare the hPTHR-induced
response to that of a system that utilizes the InsP pathway, we also expressed a cloned serotonin receptor, the
5-HT receptor, that couples to the phospholipase C
pathway(36) . Two parameters were analyzed for time course
studies of receptor-mediated membrane currents, the latency of onset
and the start-peak time (Fig. 5A). The response
profiles from the two receptors are very distinct from each other, with
the 5-HT response being a fast and robust one, and the hPTH response
showing a longer latency and a slower kinetics for start-peak time (Fig. 5, A and B). Specifically, application
of 5-HT produces an almost immediate current, whereas hPTH-induced
current does not commence until about 13 s after the hormone is added (Fig. 5B, top panel). The 5-HT current also
develops quickly, with an average start-peak time at 4 s, whereas
hPTH-induced current takes about 7 s to develop (Fig. 5B, bottom panel). These differences are
statistically significant, and they suggest that the two receptors may
use different signaling pathways.
receptor. Oocytes were clamped at -80 mV. B, relative time courses of the latency of onset and the
start-peak time between hPTH- and 5-HT-induced responses. Data are
presented by mean ± S.E.; n is for numbers of oocytes
used. The latency of onset for hPTHR, 12.8 ± 0.8, n = 52; for 5-HT, 0.6 ± 0.06, n = 14. The start-peak time for hPTHR, 6.9 ± 0.5, n = 52; for 5-HT, 3.9 ± 0.3, n = 14. * (top graph) indicates a significant
difference for the latency time between the 5-HT response and hPTH
response (p < 0.01). * (bottom graph) indicates a
significant difference for the start-peak time between the two groups (p < 0.01).
receptors were roughly comparable. The radioligand
binding studies showed 2.2 10
and 4.2
10 receptors/oocyte for hPTH and 5-HT receptors, respectively. Thus, it is unlikely that the
qualitative and quantitative differences between the PTH and the
5-HT systems are related to different levels of receptor
expression.Examination of Desensitization Properties for the Human
Parathyroid Hormone Receptor and the 5-HT
G protein-coupled pathways often display marked
desensitization, operationally defined as a reduced response to
continued or repeated stimulation of agonists. The response induced by
both the hPTHR and 5-HT Receptor receptor show strong homologous
desensitization, as the second stimulation by the same agonist produced
little or no membrane current (Fig. 6A). It has been
well characterized that 5-HT receptor uses inositol
trisphosphate as the second messenger to induce calcium release from
intracellular stores. To examine the possibility that hPTH induces
Ca release by activating the same Ca release mechanism as the 5-HT receptor, the hPTH
receptor and 5-HT receptor were coexpressed in oocytes.
When hPTH application has caused a complete homologous desensitization,
5-HT stimulation was still effective in releasing Ca (Fig. 6B). The results suggest that the
5-HT receptor and the hPTH receptor cause intracellular
calcium release through distinct pathways.
and hPTH
receptor cDNA. After the cell is completely desensitized to hPTH
stimulation, 5-HT still elicited an inward current, suggesting that the
two receptors are coupled to different signaling
pathways.
Role of Inositol 1,4,5-Triphosphate in Signaling by Human
Parathyroid Hormone Receptor
It has previously been proposed
that the cytosolic Ca signal for PTH is activated by
InsP(20) . However, some studies indicate that this
may not be the case in every system(23) , and the existence of
InsP-insensitive calcium compartments that are activated by
PTH has been proposed(22) . Our kinetic and desensitization
data also suggest that possibility, and further experiments were
designed to test it more directly by studies with microinjection of
InsP. Strong activation of the Cl channel
was observed upon initial injection of InsP, but no
response was observed with a second injection (Fig. 7, top
trace). Thus, either the InsP receptor is desensitized
or the Ca pools, which respond to injected
InsP, are emptied. 5-HT and InsP also showed
cross-desensitization as expected (Fig. 7, middle two
traces), since they share the same calcium release pathway.
However, in hPTH-desensitized cells, InsP injection still
elicited an inward current, and in cells that were unresponsive to
injected InsP, a strong signal was still observed upon
addition of PTH (Fig. 7, bottom two traces).
and still respond
to PTH. Membrane current traces were recorded at a holding potential at
-80 mV. InsP microinjected into oocytes induces an
inward current and desensitizes the same oocyte to subsequent
InsP injection (top trace). 5-HT receptor-mediated response desensitizes the oocyte to subsequent
InsP injection (second trace from top),
and InsP injection similarly desensi-tizes the cell to 5-HT
stimulation (third trace from top). However, in a
cell that is desensitized to hPTH stimulation, InsP injection still elicited a strong inward current (fourth
trace from top), and in a cell that is desensitized to
InsP treatment with PTH still elicited a strong inward
current (fifth trace).
compartments that are
insensitive to injected InsP, we examined whether PTH
signaling might proceed through the cADPR pathway (44, 45, 46) or the recently described
NAADP pathway(51) . However, we observed no
effect of injection of these compounds on the Cl current in Xenopus oocytes. In addition, as expected
from the above results, inhibitors that block both InsP and
cADPR signaling systems were found to have no affect on the PTH
responses in oocytes (data not shown). However, unexpectedly we found
that NADP, thio-NADP, and the
NADP analogue, 3-acetylpyridine-ADP,
are all strong inhibitors of the cytosolic Ca response to PTH in oocytes (Fig. 8). This inhibition
persists for a few hours, but the hormone response is eventually
regained. In control experiments, injection of these compounds alone
had no impact on cytosolic Ca, or on oocyte
responsiveness to serotonin.
inhibits
the cytosolic Ca response to hPTH but not to 5-HT or
InsP. Oocytes were voltage clamped at -80 mV. A, NADP microinjected into control oocyte; B, NADP microinjected into oocytes expressing
PTHR prior to stimulation with hPTH; the oocyte was allowed to recover
for 12 h prior to second exposure to PTH; C, NADP microinjected into oocytes expressing 5-HT receptor prior to
stimulation with 5-HT; D, response of NADP injected oocytes to InsP. Similar results were
obtained with either thio-NADP, and 3-acetyl
pyridine-ADP used in place of
NADP.
can be detected
by activation of an endogenous Cl channel.
Identification of the PTHR-stimulated signal as a Cl current was accomplished by its inhibition with niflumic acid and
by elimination of the signal through clamping the voltage at -25
mV, the known resting potential for Cl in this
system. The Ca dependence for this current is
demonstrated by its inhibition with injected EGTA, and a lack of
inhibition by external EGTA establishes that the signal is not mediated
by extracellular Ca. The results are consistent with
earlier demonstrations that the PTHR activates increases in cytosolic
Ca in wild-type and in transfected
cells(7, 12, 15, 16) . by a cAMP-mediated stimulation of a plasma
membrane channel in some cells(18) . This effect is dependent
on extracellular Ca and thus appears unlikely to be
responsible for the signals we observe. The PTHR expressed in oocytes
does activate production of cAMP(32) , and thus a lack of any
detectable affect of injected cAMP or its nonhydrolyzable analogues
represents additional evidence that our signals are not mediated
through this plasma membrane channel. The apparent lack of any effect
of cAMP on PTH responses or desensitization are also of interest since
protein kinase A has been implicated in the down-regulation of PTHR in
some cultured cell systems (33, 34) . Further work
should also examine the possible role of protein kinase C in this
system, since it has also been implicated in down-regulation of
PTHR(37) . response to PTH in Xenopus oocytes expressing the PTHR. Earlier work with mRNA extracts from
target cells for PTH demonstrated that the receptor can be expressed in
oocytes, utilizing the cAMP response to measure receptor
function(32, 38) . Thus, the two primary signaling
responses to PTH have now been demonstrated in this system. This is
significant since cell systems that express the PTHR do not always show
both responses. For example, some cultured bone cells show elevated
cAMP in response to PTH, but no detectable increases in cytosolic
Ca(14) , while cultured keratinocytes show a
cytosolic Ca response but no elevation in
cAMP(15) . Also, some forms of PTH can generate one response
but not the other (16) , and in one study, cells that were
desensitized with regard to the cAMP response still elicited a
cytosolic Ca response (17) . in PTHR signaling and the inhibition of signaling
by NADP. The most direct interpretation of our data
with regard to the role of InsP is that cytosolic
Ca signaling by PTHR in oocytes is not mediated by
this second messenger. This interpretation is supported by several
observations. First, oocytes that cannot respond to injected InsP still show a strong response to added PTH. Second, cells whose
InsP-sensitive Ca stores appear to be
depleted by activation of coexpressed 5-HT receptors still respond to
added PTH. Third, the response to PTH is consistently slower than that
to 5-HT, suggesting that signaling is through a different route, and
fourth, the PTH response is strongly inhibited by NADP and related compounds, none of which have any apparent chemical
or biological relationship to InsP. compartments that are not accessed either by injected materials
or by InsP generated by other receptors. Such compartments
could be activated by cAMP or InsP released in localized
sites near the PTHR, while remaining unresponsive to the injected
materials or to increased concentrations of these second messengers
generated by activation of other receptor systems. Although we know of
no evidence for such compartments, this possibility cannot be excluded
at this time. Thus, we cannot unequivocally conclude that InsP is not the signal for cytosolic Ca increases
triggered by the PTHR in the frog oocyte. However, at the minimum, our
studies demonstrate the existence of unique Ca compartments in oocytes that are responsive to the PTH receptor.
The unique nature of these compartments is either their
unresponsiveness or their inaccessibility to presently known signals
and their sensitivity to NADP. is probably the most widely studied signal for cytosolic
Ca, three alternative second messenger candidates are
known:
cADPR(41, 42, 43, 44, 45, 46) ,
sphingosine-phosphate(47, 48, 49, 50) ,
and NAADP(51) . None of these have yet been
shown to be active in Xenopus oocytes, and there is evidence
that the cADPR pathway does not function in this system(39) .
However, it has also been reported that acetylcholine and
thyrotropin-releasing hormone mobilize calcium from functionally
separate stores in Xenopus oocytes(40) , although the
signaling systems for these two responses have not been identified. In
the present work, we were unable to demonstrate responses of frog
oocytes to either injected cADPR or NAADP, but we have
not yet examined the sphingosine-phosphate pathway. has not been described
previously. This effect is not observed with the serotonin receptor,
which is known to utilize the InsP pathway, and it thus
provides further evidence that InsP may not be the signal
for the PTH system. It is of interest that the most sensitive assay for
PTH is based on a procedure that requires activation of
glucose-6-phosphate dehydrogenase and production of NADPH(52) .
The biochemical rationale for this assay is not understood, but our
results with NADP now provide further impetus for
examination of the possible role of this coenzyme in biochemical
actions of PTH. As indicated in Fig. 8, the inhibition of
cytosolic Ca signaling is acute and persists for
hours following injection of NADP. This effect could
be the result of direct interaction of NADP with some
regulatory element in the PTH signaling pathway or a more general
effect of a significant alteration of the NADP/NADPH
ratio in the cytoplasm and its subsequent impact on the
oxidation/reduction or catabolic/anabolic state of the cell. It may be
that the signaling by PTHR is modulated by metabolic state since this
hormone is known to produce both anabolic and catabolic affects on
different cell types under different conditions. Further study of the
regulation of signaling by the PTHR in the frog oocyte system may shed
light on these possibilities.
), inositol 1,4,5-trisphosphate; 8-Br-cAMP,
8-bromo-cAMP; 8-CPT-cAMP, 8-(4-chlorophenylthio)-cAMP; 5-HT, serotonin
(5-hydroxytryptamine); 4-AP, 4-aminopyridine; TEA, tetraethylammonium;
cADPR, cyclic adenosine diphosphate ribose; NAADP,
nicotinic acid adenine dinucleotide phosphate.
We thank Joyce Hurley and Dr. Anton Mestek for helpful
discussions.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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