From the
Neuropeptide Y (NPY) and norepinephrine, found co-localized in
sympathetic neurons innervating blood vessels, exert synergistic
responses on vasoconstriction. To examine the signaling mechanisms
involved, free of complications associated with mixed receptor
populations, we have established a stable Chinese hamster ovary cell
line expressing both Y1-NPY and
Neuropeptide Y (NPY)
Receptors for NPY are present in a wide variety of tissues including
atrial cells, coronary and cerebral blood vessels, and in aortic and
vascular smooth muscle cell lines. These correspond to at least three
receptor subtypes, designated Y1, Y2, and Y3, as identified using
receptor-specific peptide agonists
(4, 5, 6) .
Whereas the presynaptic responses are mediated chiefly through Y2
receptors, the pressor effects of NPY are due predominantly to
interaction with the Y1 species (4-6). The latter receptor has
recently been cloned from several species and revealed to be a member
of the family of G protein-coupled receptors
(7, 8) .
This is consistent with earlier functional studies, using a variety of
tissues and cell lines, which suggested that NPY receptors coupled to
second messenger systems via an interaction with one or more
PT-sensitive G proteins
(9, 10, 11, 12) .
Both Y1 and Y2 receptors couple to an inhibition of adenylate
cyclase
(4, 5, 6) . In addition, occupation of Y1
receptors has been well documented to promote a rise in
[Ca
Because of the
essential role that Ca
The current study
demonstrates that in transfected CHO cells the Y1-NPY receptor subtype
couples, via a PT-sensitive G protein, to mobilization of
Ca
In addition
to Ins(1,4,5)P
A less
well characterized Ca
NPY has been previously shown to synergize with other agonists
promoting PtdInsP
A major thrust of the current
study was directed toward determining which of the two pathways, acting
downstream of PtdInsP
Indeed our results demonstrate that PKC
activation is necessary for at least one process acting downstream of
the Y1 receptor: potentiation of the PE-stimulated activation of
PLA
In conclusion, we have used CHO cells
heterologously expressing the Y1-NPY and
Transfected CHO
cells were stimulated for 1 min with PYY (100 nM) or PE (10
µM) either alone or in combination. Results
(n=3) are expressed as percent of the unstimulated
inositol phosphate levels which averaged 58, 97, and 50 cpm for
Ins(1,4)P
We thank Vikki Falls and Yvonne Hort for technical
assistance, Robert Graham for helpful advice and generous provision of
the
-adrenergic
receptors. Occupation of either receptor species, with 100 nM
peptide YY (PYY) or 10 µM phenylephrine (PE),
respectively, resulted in a rapid increase in the cytoplasmic free
calcium concentration
([Ca
]
) as assessed
with Fura-2/AM. The rise due to PYY, but not that due to PE, was
abolished by pretreatment with pertussis toxin. Both responses were
largely maintained in the absence of extracellular
Ca
, but abolished by prior depletion of intracellular
Ca
pools with either thapsigargin or
2,5-di-(t-butyl)-1,4-benzohydroquinone. Using cells prelabeled
with myo-[
H]inositol, PE promoted a
rapid (5 s) rise in inositol 1,4,5-trisphosphate
(Ins(1,4,5)P
) as analyzed by anion-exchange high pressure
liquid chromatography, whereas the response to PYY (first significant
at >15 s post-stimulation) was too slow to play a causative role in
Ca
mobilization. Combination of PE and PYY resulted
in increases in [Ca
]
which were at best additive, whereas they promoted a clearly
synergistic rise in Ins(1,4,5)P
at both 15 and 60 s.
Co-stimulation also resulted in a synergistic activation of both
protein kinase C (PKC) and [
H]arachidonic acid
release. In either instance PYY alone was without effect. The
potentiation of arachidonic acid release was abolished by depletion of
cellular PKC following chronic treatment with phorbol esters. It is
suggested that the ability of PYY to mobilize Ca
in
an Ins(1,4,5)P
-independent fashion minimizes the functional
importance of the capacity to potentiate PE-stimulated
Ins(1,4,5)P
generation. Instead the major conseqences of
the synergistic activation of phospholipase C are mediated via PKC, the
other route of the signaling pathway.
(
)
is an abundant
peptide hormone and neurotransmitter that is important in mediating
anxiety responses and the regulation of food intake. It also plays a
major role in the control of cardiovascular function and vascular tone,
acting through both the peripheral and central nervous
systems
(1, 2, 3, 4) . The presence of
NPY at high concentrations in the nerve terminals surrounding blood
vessels, particularly arteries, suggests that it influences vascular
resistance and tissue perfusion. NPY acts through both pre- and
postsynaptic mechanisms on the heart and cardiovascular system.
Presynaptically the peptide exerts long lasting inhibitory effects on
the release of norepinephrine and acetylcholine by sympathetic and
parasympathetic nerves. At the postsynaptic site, NPY has been shown to
act as a potent vasoconstrictor in its own right, as well as to
potentiate the constrictor effects of numerous pressor agents,
including norepinephrine, with which it is found co-localized in
sympathetic neurons
(1, 2, 3, 4) .
]
in vascular
smooth muscle cells
(11, 13) , human erythroleukemia
(HEL) cells
(10, 14) , and neuroblastoma SK-N-MC
cells
(15) . The coupling of a single NPY receptor species to the
two intracellular signaling systems has been confirmed by heterologous
expression of the Y1 receptor
(7, 8) .
plays in the regulation of
vasoconstriction
(16, 17) , the effects of NPY on
[Ca
]
are likely to be
more important in that regard than those on cAMP. These NPY-induced
Ca
responses involve mobilization of Ca
from intracellular stores and a consequent Ca
influx through non-voltage-gated channels (7, 8, 10, 11, 13, 14).
By far the best understood means of linking Ca
mobilization with G protein-coupled receptors is via the
activation of PtdInsP
hydrolysis and release of the
intracellular messengerIns(1,4,5)P
(18). This reaction,
catalyzed by PLC, also liberates diacylglycerol, the endogenous
activator of PKC
(19) . However, the mechanism underlying the
Y1-NPY receptor-stimulated rise in
[Ca
]
remains unclear
with Ins(1,4,5)P
being clearly implicated in only one
(14) of a number of
studies
(10, 11, 12, 13, 20) . In
addition, there is very little understanding of the signaling pathways
through which Y1-NPY and
-adrenergic receptors
interact synergistically to promote vasoconstriction. A potentiation of
PLC activation by NPY has been reported in some
(20, 21) but not all
(13) studies. Furthermore, in
experiments with arterial preparations, a requirement for
Ca
influx through voltage-gated Ca
channels has sometimes
(22, 23) but not
always
(24, 25) been reported. These discrepancies might
reflect drawbacks inherent in the use of arterial preparations which
contain mixed populations of both cell type and receptor species. For
example, in rabbit aorta,
-adrenergic receptors
couple predominantly to Ca
influx, whereas occupation
of
-receptors causes a phasic contraction associated
with PtdInsP
hydrolysis and Ca
mobilization (26). To obviate these difficulties we sought to
explore some of the mechanisms underlying the synergistic interactions
of NPY and PE using a defined model system. This consisted of a single
cell type (CHO cell) in which the Y1-NPY and
-adrenergic receptor subtypes were heterologously
expressed.
Materials
All media and materials for tissue
culture were purchased from Cytosystems, Castle Hill, NSW, Australia,
and radiochemicals from DuPont NEN. NPY and its related peptides, as
well as peptides for PKC assays, were obtained from Auspep Pty. Ltd.,
Parkville, Victoria, Australia. HPLC columns and P81 phosphocellulose
paper were from Whatman. TLC plates were from Merck. Fura-2/AM was
supplied by Molecular Probes, Eugene, OR. All other biochemicals were
from Sigma or Boehringer Mannheim.
Cell Culture and Transfection
Cells (CHO
K1:ATCC:CCL 61) were maintained in 5% CO in
Dulbecco's modified Eagle's medium/Ham's F-12 medium
with 2 mM glutamine, 100 IU of penicillin, streptomycin at 100
µg/ml, and 10% fetal calf serum. CHO cells were co-transfected
using a modified calcium phosphate method with the hamster
-adrenergic receptor cDNA in a pMT2 vector
(27) and the human Y1-NPY receptor cDNA in the pcDNA Neo
vector
(7) . Stably transfected cell populations were selected
with neomycin (G418). Cells were harvested by treatment with 0.2% EDTA
in PBS and immediately used in the binding assay or functional assays
described.
Binding Assay
Stable transfected cells were
selected and assayed for their ability to bind radiolabeled PYY and
prazosin, an antagonist at the -adrenergic receptor.
PYY and the Y1 receptor-specific ligand
NPY(Leu
,Pro
) were used to compete for the
binding of
I-labeled PYY, and PE for the binding of
[
H]prazosin. Transfected cells were harvested
with EDTA/PBS and washed twice in the assay buffer (50 mM
Tris-HCl, pH 7.4, 2 mM CaCl
, 5 mM KCl,
120 mM NaCl, 1 mM MgCl
, and 0.1% bovine
serum albumin). Washed cells (1
10
) were incubated
in 0.5 ml of assay buffer in the presence of labeled agonist and
increasing concentrations of competitors for 1 h at room temperature.
After centrifugation for 4 min in a microcentrifuge, the tips of the
tubes containing the pellets were cut off and placed in plastic tubes.
Radioactivity associated with the pellets was counted for 1 min in a
gamma counter. Receptor numbers were calculated as 17,900 and 19,000
per cell for Y1-NPY and
-adrenergic receptors,
respectively. Measurement of
[Ca
]
-Cells
expressing the human Y1-NPY and hamster
-adrenergic
receptors were suspended in loading medium (modified RPMI, 10
mM Hepes, 1% newborn fetal calf serum) and incubated in a
spinner flask at 37 °C for 2.5 h at 10
cells/ml. Cells
were then treated with 1 µM Fura-2/AM for 30 min at 37
°C, washed twice with loading medium, and resuspended at 5
10
cells/ml. Immediately prior to use, an aliquot of 2
10
cells was resuspended in 2 ml of modified KRB
buffer (135 mM NaCl, 4.7 mM KCl, 1.2 mM
MgSO
, 1.2 mM KH
PO
, 5
mM NaHCO
, 1 mM CaCl
, 2.8
mM glucose, and 10 mM Hepes, pH 7.4) containing 1
µM sulfinpyrazone (28). Fluorescence recordings were made
on a Hitachi fluorescence spectrometer (F4010) at 340 nm (excitation)
and 505 nm (emission) with slit widths of 5 nm and a response time of 2
s. All fluorescence traces are representative of at least three
separate experiments and were calibrated as described previously
(28) using the published formulas (29).
Measurement of Inositol Phosphate Production
Cells
were labeled with myo-[H]inositol (10
µCi/ml) for 48 h under culture conditions, then harvested and
transferred to spinner culture as described above. After 2 h they were
washed and resuspended at 2
10
cells/ml in 0.5-ml
aliquots of modified KRB buffer containing 0.1% bovine serum albumin.
Experimental additions were made as doubly concentrated stock solutions
in 0.5 ml of the same buffer. Reactions were stopped with the addition
of 100% trichloroacetic acid (100 µl), and the solutions were
vortexed and left on ice for 20 min. After centrifugation for 4 min in
a microcentrifuge, the supernatant was removed and extracted three
times with 5 ml of diethyl ether. After removal of cations with Dowex
50W-X8, inositol phosphates in the aqueous extracts were separated and
analyzed by anion-exchange HPLC using a Partisphere PAC column eluted
with ammonium phosphate (pH 3.8). The exact gradient and relative
elution times of Ins(1,3,4)P
and Ins(1,4,5)P
were exactly as described previously
(28) . Eluted
fractions (0.5 ml) were mixed with 4 ml of scintillant, and
radioactivity was determined by liquid scintillation spectrometry. Measurement of [
H]Arachidonic Acid
Release-Cells (10
/ml) were labeled with
[
H]arachidonic acid (1.3 µCi/ml) for 3 h at
37 °C in spinner culture. They were then washed, resuspended, and
incubated with experimental additions exactly as described for the
inositol phosphate studies. After incubation the experimental tubes
were chilled on ice, and the cells then pelletized using a
microcentrifuge. Supernatant (0.5 ml) was transferred to a
scintillation tube with 5 ml of scintillant, and radioactivity was
determined. For confirmation of the release of arachidonic acid,
incubations were stopped with the addition of 1.9 ml of ice-cold
chloroform/methanol/HCl (100:200:2, v/v), and lipids were extracted
with further addition of chloroform and H
O. Additional
arachidonic acid (5 µg) was added to aid in recovery during the
extractions. The lower lipid-containing phase was dried under nitrogen,
resuspended in chloroform, and applied to thin layer chromatography on
Silica Gel 60 F
20
20-cm, 0.2-mm thick TLC plates
in ethyl acetate:isooctane:acetic acid:H
O (9:5:2:1) or
benzene:chloroform:methanol (80:15:5). Free arachidonic acid was
identified by comigration with a known standard and staining with
iodine crystals or Coomassie Blue stain, and the appropriate spot was
scraped from the plate and quantified by scintillation counting.
Measurement of PKC Activity
CHO cells
(approximately 2 10
) were plated out 24 h prior to
the experiment in 9.6 cm
wells. Medium was aspirated, and
the cells were washed with 2 ml of PBS. Incubations were carried out in
1 ml of modified KRB buffer with either 100 nM PYY, 10
µM PE, both agonists or vehicle. After 15 s, medium was
aspirated, and the cells were washed once with ice-cold PBS and scraped
into 1.5-ml centrifuge tubes with 0.4 ml of homogenization buffer (20
mM Tris-HCl, pH 7.5, 2 mM EDTA, 0.5 mM EGTA,
1 mM dithiothreitol, 200 µg/ml (w/v) leupeptin, 2
mM benzamidine, and 2 mM phenylmethylsulfonyl
fluoride). The cells were extracted by three rapid freeze-thaw cycles,
and the extracts were subjected to centrifugation at 100,000
g for 30 min at 4 °C in a Beckman 70.1 Ti rotor, to give
cytosolic and particulate fractions. The particulate fraction was
further extracted, after briefly rinsing with ice-cold PBS, by
resuspension in 0.2 ml of homogenization buffer containing 0.1% Triton
X-100. Following centrifugation, the 100,000
g supernatant from this suspension, corresponding to the original
membrane associated PKC, was assayed for activity. Samples (10 µl)
were assayed in a final volume of 50 µl, containing 24 mM
MOPS, pH 7.5, 0.04% Triton X-100, 1 mM CaCl
, 120
nM cyclic AMP-dependent protein kinase inhibitor peptide
(rabbit sequence), 0.5 mg/ml histone HIIIS, 100 µM
[
-
P]ATP (150 cpm/pmol), and 5 mM
magnesium acetate, in the absence or presence of 125 µg/ml
phosphatidylserine and 2.5 µg/ml dioctanoylglycerol. Lipids, at 5
mg/ml in chloroform/methanol (19:1), had been dried under nitrogen and
sonicated into 100 mM MOPS (pH 7.5), 1% Triton X-100 until
clear before addition to the assay buffer. After 10 min at 30 °C,
assays were terminated with the addition of 10 µl of 150
mM unlabeled ATP, and samples were spotted onto P81 paper,
washed with orthophosphoric acid, and counted for Cerenkov
radiation
(30) . PKC was determined as the additional activity
observed in the presence of the lipid activators.
Statistics
All results are presented as mean
± S.E. of the mean. Statistical significance was assessed using
Student's t test for unpaired data.
RESULTS
NPY and Adrenergic Receptor-induced Increases in
[Ca]
-Y1R-
1BAR/CHO
K1 cells, stably expressing the Y1-NPY and
-adrenergic receptors, were loaded with the
calcium-sensitive dye Fura-2/AM. In all of the following experiments
PYY, which is less hydrophobic than NPY, was used as a selective
agonist for the Y1-NPY receptor
(7) . Consistent with earlier
studies
(7) , PYY (100 nM) caused a transient increase
in [Ca
]
in CHO K1
cells expressing the Y1-NPY receptor (Fig. 1a). This was
largely unaffected by the removal of extracellular Ca
and is therefore primarily due to the mobilization of
Ca
from intracellular stores
(Fig. 1b). Occupation of the
-adrenergic receptor with 1 µM PE also
resulted in an immediate increase in
[Ca
]
, but this was
followed by only a slight decline from the peak level. Extracellular
Ca
was essential for the sustained phase
(Fig. 1b), suggesting that it was due to Ca
influx. Y1R-
1BAR/CHO cells pretreated with PT (200 ng/ml)
were barely responsive to PYY, whereas both Ca
mobilization and Ca
influx due to PE were
virtually unaffected (Fig. 1, c and d). This
suggests that the Y1 and
-adrenergic receptors couple to rises in
[Ca
]
via distinct G
proteins, which show differential sensitivities to PT.
Figure 1:
Calcium
responses in CHO cells expressing Y1-NPY and
-adrenergic receptors. After loading for 30 min with
1 µM Fura-2/AM, cells were washed and resuspended at 2.0
10
cells/ml in KRB buffer. Fluorescence was
measured using cells maintained under continuous stirring in a cuvette
at 37 °C. For further details, see ``Experimental
Procedures.'' Changes in [Ca
] were
measured using cells incubated in the presence (Panels a and
c) or absence (b and d) of 1 mM
extracellular CaCl
and that had been pretreated overnight
with 200 ng/ml pertussis toxin (c and d) or not
(a and b). Gaps in the recordings are due to the
opening of the compartment to make additions. Arrows mark the
point of addition of PYY (100 nM) or PE (1
µM).
The next
series of experiments were undertaken to examine the interactive
effects of PYY and PE on
[Ca]
. This was tested
in Fura-2/AM loaded Y1R-
1BAR/CHO cells using three different
experimental protocols: co-addition of the two receptor agonists; prior
addition of PE followed by PYY; or addition of PYY first followed by
PE. The results of these experiments are summarized in Fig. 2.
Co-stimulation with PYY (100 nM) and PE (100 nM or 1
µM) resulted in
[Ca
]
increases which
were not significantly greater than the sums of the responses to both
agonists alone (top panel). Using the alternative experimental
protocols, no effect of pretreating the cells with 1 µM PE
was seen on the subsequent rises in
[Ca
]
induced by a
range of PYY concentrations (middle panel); nor did prior
addition of PYY (100 nM) affect the responses to various doses
of PE (bottom panel). Taken together these results suggest
that PYY and PE exert effects on
[Ca
]
which are at best
additive, but clearly not synergistic.
Figure 2:
Effect of co-stimulation of Y1-NPY and
-adrenergic receptors on
[Ca
] in transfected CHO cells. Cells were
loaded with Fura-2/AM and [Ca
] measured as
described in the legend to Fig. 1. Top panel, average
increases in [Ca
] in cells stimulated with
PYY (100 nM) either alone, or in the combined presence of PE
(100 nM or 1 µM). Middle panel,
dose-dependence of average increases in
[Ca
] versus PYY concentration in
cells that had, or had not, been prestimulated with 1 µM
PE. Where appropriate, PYY was added as soon as the PE response had
plateaued out. Bottom panel, dose dependence of average
increases in [Ca
] versus PE
concentration in cells that had, or had not, been prestimulated with
100 nM PYY. Each column or data point represents the average
of duplicate determinations from at least three
experiments.
Y1-NPY Receptor-stimulated PLC Activity and Potentiation of
Adrenergic Receptor PLC Stimulation
Mobilization of
Ca from intracellular stores occurs most frequently
as a result of Ins(1,4,5)P
generation
(18) . When
added to Y1R-
1BAR/CHO cells, prelabeled with
myo-[
H]inositol, PE (10 µM)
induced an increase in Ins(1,4,5)P
which was rapid in onset
(5 s) and sustained for at least 5 min (Fig. 3). In marked
contrast, the rise in Ins(1,4,5)P
stimulated by PYY was not
significantly different from control until 60 s after addition. This
demonstrates clear differences in the mechanisms by which the
-adrenergic and Y1 receptors couple to PLC. Most
importantly, the rise in Ins(1,4,5)P
seen under these
conditions appears to be too slow to explain the PYY-stimulated
Ca
mobilization which peaks within 10-15 s (see
Fig. 1
)
Figure 3:
Time courses of Ins(1,4,5)P
generation in transfected CHO cells. Cells prelabeled for 48 h with
[
H]inositol were stimulated for various times
with either PYY (100 nM) or PE (10 µM). Inositol
phosphates in cell extracts were analyzed by anion-exchange HPLC. For
further details, see ``Experimental Procedures.'' Each point
represents the average of at least duplicate determinations from two
(PE) or three (PYY) independent experiments. The earliest significant
rises occurred at 5 s (p < 0.02) and 60 s (p <
0.005) for PE and PYY, respectively. Results are expressed as a
percentage of the zero time control which averaged 69 cpm (n = 8).
We next examined the effects of combined addition of
PYY and PE on PLC activity (Fig. 4). One minute of stimulation
with both agonists raised Ins(1,4,5)P to a level which was
approximately 1.4-fold higher than simple summation of the individual
responses. This potentiation was even more apparent at the 15 s time
point where PYY was unable to exert any significant effect by itself
but almost doubled the response to PE (p < 0.05 versus PE alone). This was a true potentiation of PLC activity, rather
than an inhibition of Ins(1,4,5)P
degradation, since the
latter's metabolites, Ins(1,3,4)P
and
Ins(1,4)P
, were also augmented under these conditions
(). The potentiation of PE-stimulated Ins(1,4,5)P
generation by PYY is noteworthy in view of the fact that the
Ca
responses, under corresponding circumstances, were
no more than additive (cf. Fig. 2). This is most
probably explained by the ability of PYY to generate a robust
Ca
mobilization independently of
Ins(1,4,5)P
, which would tend to minimize the functional
relevance of the potentiation of Ins(1,4,5)P
generation.
Figure 4:
Effect of combined addition of PE and PYY
on Ins(1,4,5)P generation. Transfected CHO cells were
stimulated for either 15 s or 1 min in the presence of PYY (100
nM) and PE (10 µM) either alone or in
combination. Ins(1,4,5)P
was measured as described in the
legend to Fig. 3 and under ``Experimental Procedures.'' Each
column represents the average of at least duplicate determinations from
two independent experiments. Results are expressed as a percentage of
the control response which averaged 56 and 50 cpm at 15 s and 1 min,
respectively.
The simplest explanation for the observed potentiation of PLC
activity would be that it is secondary to PYY-induced Ca mobilization. To investigate this possibility intracellular
Ca
pools were depleted by sequential addition of the
chelating agent EGTA and the Ca
-ionophore ionomycin.
Under these conditions the capacity of either PYY or PE to raise
[Ca
]
was completely
blocked (Fig. 5, upper panel). Although slightly
attenuated as compared to the response in non-depleted cells, addition
of PE alone to Ca
-depleted cells still resulted in a
2.5-fold stimulation of PtdInsP
hydrolysis (Fig. 5,
lower panel). Similarly, PYY increased PE-stimulated
Ins(1,4,5)P
production from approximately 260% of control
to 400% in the depleted cells, as compared to a rise from 290 to 480%
in the non-pretreated cells. Therefore, the synergistic activation of
PLC is only partially inhibited under circumstances in which the rise
in [Ca
]
is abolished.
This indicates that, although the PYY-induced Ca
mobilization might, under normal circumstances, contribute to the
potentiation of PLC activity, it is not the primary mechanism
underlying that potentiation.
Figure 5:
Effect of Ca depletion
on the potentiation of PE-stimulated Ins(1,4,5)P
generation
by PYY. Transfected CHO cells were preincubated in KRB medium for 10
min and then stimulated with PE (10 µM) in the presence or
absence of PYY (100 nM) for 15 s. Cells depleted of
Ca
were treated during the preincubation for 3 min
with 2 mM EGTA, and then for a further 4 min with 100
nM ionomycin, prior to addition of the receptor agonists.
Upper panel, representative [Ca
]
trace showing depletion of intracellular stores. Lower panel,
Ins(1,4,5)P
responses in non-depleted and
Ca
-depleted cells. Ins(1,4,5)P
was
measured as described in the legend to Fig. 3 and under
``Experimental Procedures.'' Each column represents the
average of duplicate determinations from one experiment representive of
two.
Further Characterization of Y1-NPY Receptor-coupled
Ca
Because an increase in
Ins(1,4,5)P Mobilization
does not appear to account for the rise in
[Ca
]
due to PYY, we
next sought evidence for the involvement of either cyclic ADP-ribose
(31) or sphingosine 1-phosphate
(32) , both recently
proposed Ca
mobilizing agents (Fig. 6). Cyclic
ADP-ribose has been shown to act on those Ca
stores,
present in certain cell types, which are depleted by ryanodine and
caffeine
(33) . However, these agents were without effect in CHO
cells when added either individually (not shown) or in combination
(Fig. 6b). This suggests that the appropriate stores are
not present and, hence, that cyclic ADP-ribose is unlikely to be a
mediator of the Ca
mobilization due to PYY in CHO
cells. Sphingosine 1-phosphate is a metabolite of sphingosine formed in
fibroblasts in response to growth factor stimulation
(34) . When
added to permeabilized fibroblasts it brings about a rapid release of
Ca
from a non-mitochondrial store
(35) .
Addition of DL-threo-erythro-sphingosine, a
competitive inhibitor of sphingosine 1-phosphate formation
(34) ,
to the Y1R-
1BAR/CHO cells resulted in complex effects on
[Ca
]
: an initial
decrease, followed by a transient rise (Fig. 6c).
However, the inhibitor exerted no effect on the ability of PYY to raise
[Ca
]
, suggesting that
formation of sphingosine 1-phosphate was not involved in the mediation
of that response (Fig. 6c).
Figure 6:
Evidence
against the involvement of cyclic ADP-ribose and sphingosine
1-phosphate in the mobilization of intracellular Ca
by PYY. Measurements of [Ca
] in transfected
CHO cells were performed as described in the legend to Fig. 1 and under
``Experimental Procedures.'' Arrows mark the point
of addition of 100 nM PYY, 50 µM ryanodine, 5
mM caffeine, and 10 µMDL-threo-erythro-sphingosine.
Because PE and PYY appear
to act via different mechanisms, we reasoned that it might be possible
to distinguish between the intracellular stores from which they release
Ca. The fungal alkaloid thapsigargin acts as an
inhibitor of certain intracellular calcium ATPases and has been widely
used to deplete Ins(1,4,5)P
-sensitive Ca
pools
(36) . Another compound, tBuHQ, acts through a
similiar mechanism but is possibly more selective than thapsigargin in
terms of the stores upon which it acts (37). When added to CHO cells,
both thapsigargin and tBuHQ caused a transient rise in
[Ca
]
, after which
neither PYY nor PE were effective, suggesting that the PYY and PE
responses are functionally indistinguishable at this level
(Fig. 7). This finding, together with the demonstrated absence of
a caffeine-sensitive store in CHO cells (Fig. 6b),
suggests that PYY and PE might mobilize Ca
from the
same intracellular pool, albeit using different mechanisms.
Figure 7:
Depletion of the PYY and PE-sensitive
intracellular Ca pools by thapsigargin and tBuHQ.
Measurements of [Ca
] in transfected CHO
cells were performed as described in the legend to Fig. 1. and under
``Experimental Procedures.'' Arrows mark the point
of addition of 100 nM PYY, 1 µM PE, 100
nM thapsigargin, and 20 µM
tBuHQ.
Y1-NPY Receptor-mediated Augmentation of Adrenergic
Receptor-stimulated Protein Kinase C Activity
Because of the
results described above, which down-played the functional relevance of
the synergistic increase in Ins(1,4,5)P generation during
co-stimulation with PYY and PE, it was important to determine whether
the potentiation of PtdInsP
hydrolysis might be manifest as
a potentiation of PKC activity, the other arm of the signaling pathway.
As shown in Fig. 8, a 15-s stimulation with PE-increased
membrane-associated PKC activity in Y1R-
1BAR/CHO cells by
approximately 20% (p < 0.05), whereas PYY alone exerted no
statistically significant effect (p > 0.1). However, in the
presence of PYY, the PE response was augmented by almost threefold
(p < 0.005 versus PE alone). This clearly
demonstrates that the potentiation of PLC activity due to
co-stimulation with PE and PYY does have functional significance in
terms of the activation of PKC, if not with respect to the mobilization
of Ca
.
Figure 8:
Effects of PYY and PE on PKC activity.
Transfected CHO cells were incubated for 15 s with PPY (100
nM) and PE (10 µM) either alone or in
combination. They were then extracted, and particulate fractions were
prepared and assayed for PKC activity as described under
``Experimental Procedures.'' PKC activity is expressed as a
percentage of unstimulated control which corresponded to 482 pmol/min
phosphotransferase activity per mg of protein. Results were normalized
using background activity observed in the absence of lipid activators
and are means of six independent experiments, each assayed in
triplicate.
Y1-NPY Receptor-mediated Augmentation of Adrenergic
Receptor-stimulated PLA
In addition to
rises in [Ca Activity
]
and PKC
activity, an increase in arachidonic acid, generated by action of the
enzyme PLA
, has also been proposed as an important event in
the regulation of vasoconstriction
(16) . Furthermore, receptors
coupling through PT-sensitive G proteins have previously been shown to
stimulate PLA
activity in CHO cells
(38) . We
therefore sought to determine whether this was also the case for PYY.
Using Y1R-
1BAR/CHO cells prelabeled with
[
H]arachidonic acid, PYY alone was unable to
stimulate PLA
activity (Fig. 9, top). In
contrast PE stimulated arachidonic acid release by approximately 80%
above basal, and this response was significantly augmented (p < 0.001) in the presence of PYY (Fig. 9, top).
Using cells pretreated with PT, PE-stimulated PLA
activity
was slightly enhanced, but PYY was no longer able to potentiate the PE
response (Fig. 9, top). This potentiation of arachidonic
acid release was due to an increase in the maximal response rather than
in sensitivity to PE (Fig. 9, bottom).
Figure 9:
Effects of PE and PYY on arachidonic acid
production. Transfected CHO cells were prelabeled for 3 h with
[H]arachidonic acid, washed, and resuspended at
10
cells/ml in KRB buffer. Arachidonic acid released into
the medium over 15 min was measured in response to PE (10
µM) or PYY (100 nM) either alone or in
combination. For further details, see ``Experimental
Procedures.'' Each column or data point represents the average of
four independent experiments each assayed in triplicate. Upper
panel, cells were cultured overnight in the presence or absence of
200 ng/ml pertussis toxin. Results are expressed as a percentage of the
basal release which averaged 4688 and 4323 cpm (n = 12)
for untreated and PT-pretreated cells, respectively. Lower
panel, dose dependence of arachidonic acid release versus PE concentration in the presence or absence of PYY (100
nM). Results are expressed as a percentage on the incremental
response due to 10 µM PE which averaged 7211 cpm (n = 12).
NPY Receptor-mediated Augmentation of Adrenergic
Receptor-mediated Arachidonic Acid Production Is Secondary to PKC
Activation
To determine the mechanism underlying the
potentiation of PE-induced arachidonic acid production by PYY,
Y1R-1BAR/CHO cells were pretreated overnight with the phorbol
ester TPA, to deplete them of PKC activity. As shown in Fig. 10,
the ability of PE alone to generate arachidonic acid was partially
impaired in the depleted cells indicating that, under normal
circumstances, the PE response is mediated as a result of both PKC
activation and the direct coupling of the receptor to PLA
.
However, the capacity of PYY to potentiate the arachidonic acid
production initiated by PE was entirely abolished in PKC-depleted
cells.
Figure 10:
Effect of the down-regulation of PKC
activity on the synergistic activation of arachidonic acid release by
PYY and PE. Transfected CHO cells were cultured overnight in the
presence or absence of 1 µM TPA and then labeled with
[H]arachidonic acid as described in the legend to
Fig. 9. Arachidonic acid released into the medium over 2 min was
measured in response to PE (10 µM) or PYY (100
nM) either alone or in combination. For further details, see
``Experimental Procedures.'' Results are expressed as a
percentage of basal which averaged 3308 and 2818 cpm (n = 9) in the untreated and TPA pretreated cells
respectively. Each column or data point represents the average of three
independent experiments each assayed in
triplicate.
DISCUSSION
In many blood vessels the pressor actions of PE, acting
through -adrenergic receptors, are potentiated by
NPY
(1, 2, 3, 4) . The aim of the current
study was to examine the mechanisms underlying this potentiation using
a model system in which the Y1-NPY and
-adrenergic
receptors were heterologously co-expressed in CHO cells. The unique
advantage of the current system is that it avoids potentially confusing
results due to the presence of multiple subtypes of NPY or adrenergic
receptors. Moreover, the fact that our findings are consistent with,
but greatly clarify and extend, those obtained using other models of
NPY receptor signaling, suggests that their discussion in the context
of vasoconstriction is warranted. However, it should be stressed that
additional mechanisms are almost certainly operative in vivo where the integration of signals from multiple receptor species
and mixed cell populations would be involved.
from a thapsigargin-depletable intracellular
store. This is consistent with results obtained using a variety of
cells expressing endogenous Y1-NPY
receptors
(10, 11, 15, 39, 40) .
However, the role of Ins(1,4,5)P
in this process has been
controversial, and a clear resolution of this controversy is an
important consequence of the present study. Although NPY has been shown
to stimulate PtdInsP
hydrolysis
presynaptically
(5, 6) , the only evidence implicating Y1
receptors in this process was obtained in a single study using HEL
cells
(14) . This was inconsistent with an earlier study using
the same cells
(10) as well as experiments performed on pig
splenic tissue
(41) , vascular smooth muscle cells (13), porcine
cultured aortic smooth muscle cells
(12) , rabbit pulmonary
artery
(42) , rat tail artery
(21) , and SK-N-MC
cells
(12, 15) . While it is possible that species,
tissue, or receptor subtype differences might be involved, the simplest
explanation for these discrepancies is methodological: in the earlier
study supporting a role for Ins(1,4,5)P
its levels were
measured by anion-exchange HPLC
(14) ; in most of the negative
reports inositol phosphates were measured by cruder chromatographic
techniques unable to resolve individual isomers. In one study a mass
assay was used, but time points longer than 60 s were not
investigated
(11) . In our hands the stimulation of inositol
phosphates, due to PYY alone, and the potentiation of the PE responses
were not seen if the cell extracts were simply analyzed by batch
elution from Dowex anion-exchange columns (not shown). We can thus
conclude that PYY clearly does stimulate PtdInsP
hydrolysis, albeit weakly. However, analysis of the time courses
of Ins(1,4,5)P
generation further revealed that the PYY
response was much slower in onset than that due to PE and is therefore
probably not coupled closely to occupation of the Y1 receptor. The
process involved in this delayed activation of PtdInsP
hydrolysis is unknown, but is not simply a consequence of the
rise in [Ca
]
, since
the Ca
mobilizing agent thapsigargin was unable to
stimulate PLC in these cells (not shown). More importantly, the delay
in the rise in Ins(1,4,5)P
clearly precludes involvement of
this second messenger in the Y1 receptor-mediated mobilization of
intracellular Ca
stores which, like the response to
PE, peaked within several seconds of agonist addition.
, a number of other molecules potentially
involved in the release of Ca
from intracellular
pools have been put forward in recent years. The most prominent of
these is cyclic ADP-ribose which appears to act through those
intracellular Ca
channels that bind the
pharmacological agent, ryanodine. These channels, intimately involved
in the process of Ca
-induced Ca
release, were originally described as components of the
sarcoplasmic reticulum, but are now known to be present on the
delimiting membranes of intracellular Ca
pools in a
number of non-muscle cell types
(18) . The cells used in the
current study did not appear to contain ryanodine-sensitive
Ca
stores, which is consistent with an earlier study
reporting the lack of immunologically detectable ryanodine receptor
protein in CHO cells
(43) . These results would therefore argue
against the involvement of cyclic ADP-ribose in mediating the
Ins(1,4,5)P
-independent Ca
mobilization
initiated by occupation of the Y1 receptor. It should be mentioned that
this conclusion is in apparent conflict with that of a previous study,
using HEL cells, in which it was shown that NPY mobilizes
Ca
from a ryanodine-sensitive store
(40) . This
conclusion was based on the observation that NPY promoted a 20% higher
rise in [Ca
]
after
prior addition of ryanodine than in its absence. Strictly speaking
these results could be explained by a ryanodine-sensitive reuptake of a
small portion of the released Ca
. The store
responsible for this reuptake need not be the same as that from which
the Ca
was released. The results presented here
clearly indicate that a ryanodine-sensitive Ca
store
is not necessary for the demonstration of Ca
mobilization following occupation of Y1-NPY receptors.
-mobilizing agent is sphingosine
1-phosphate, which has been implicated in the proliferative responses
of growth factors in fibroblasts
(32, 35) . These
responses were blocked by
DL-threo-erythro-sphingosine, a competitive inhibitor
of sphingosine 1-phosphate formation
(34) . However, this
inhibitor was without effect on the Ca
response to
PYY in CHO cells. Another candidate to be ruled out was arachidonic
acid, which releases Ca
from certain cell types
in vitro(44) . However, the concentration of
arachidonic acid in CHO cells was not increased by addition of PYY.
Therefore the coupling of Y1-NPY receptors to intracellular
Ca
mobilization does not appear to involve
Ins(1,4,5)P
, cyclic ADP-ribose, sphingosine 1-phosphate, or
arachidonic acid, and hence the underlying mechanism remains obscure.
hydrolysis in aortic smooth muscle cells
(20) and segments of rat tail arteries
(21) , but not rabbit
pulmonary artery vascular smooth muscle
(13) . As discussed above
these differences may be methodological or related to differential
expression of NPY receptor subtypes. Our results clearly demonstrate
that occupation of the Y1-NPY receptor does potentiate the activation
of PLC by PE. This occurs within 15 s of agonist addition and is
therefore more rapid in onset than the rise in Ins(1,4,5)P
due to PYY alone. The synergistic response was still apparent in
Ca
-depleted cells, arguing against the possibility
that it might be explained by a potentiation of PLC activity secondary
to PYY-induced Ca
mobilization. The mechanism
underlying this phenomenon is therefore unclear, but probably involves
some sort of cross-talk between the Y1 and
-adrenergic
receptors. This is not uncommon between two classes of receptor where,
as is the case here, one couples to PLC most probably via the
subunit of Gq/11, and the other interacts with a PT-sensitive G
protein
(28, 45) . Potentiation of PtdInsP
hydrolysis under those circumstances has been shown to require
continued occupation of both receptor species, suggestive of a dynamic
interaction occurring early in the process of receptor/effector
coupling rather than, for example, simple covalent modification of one
of the signaling molecules (28). The most obvious site for cross-talk
would be at the level of the G proteins, and it is therefore noteworthy
that the widely expressed PLC
3 isoform has been shown to be
activated in vitro by both
q/11, and
subunits
potentially associated with PT-sensitive G protein
subunits
(46, 47) .
hydrolysis, is potentially the more
important in mediating the physiological consequences of the
synergistic interaction between NPY and PE. This has not been directly
addressed previously. Several findings suggest that PKC activation,
rather than Ca
mobilization, is the more important in
this instance. First, no significant potentiation of receptor-induced
increases in [Ca
]
was
observed in the transfected CHO cells, despite potentiation at the
level of Ins(1,4,5)P
accumulation. Indeed, the
Ca
responses were no more than additive. This
apparent discrepancy is most probably explained by the capacity of PYY
to mobilize Ca
in a manner independent of
Ins(1,4,5)P
production. This separate effect of PYY would
tend to minimize the functional importance of the potentiation of
PE-stimulated Ins(1,4,5)P
generation. Second, and in
contrast to the lack of synergism in the observed Ca
responses, occupation of Y1 receptors clearly did potentiate the
PE-stimulated activation of PKC. This extends a previous study using
cultured aortic smooth muscle cells, in which NPY enhanced the
phosphorylation of an endogenous PKC substrate, but only in the
presence of a primary agonist (angiotensin II) coupled to PLC
activation
(20) . Since no Ca
responses were
measured in that study, no conclusions concerning the relative
importance of the two pathways in mediating the synergistic response
could be made. This is probably due to the fact that, until recently,
the obvious importance of a rise in
[Ca
]
for the
regulation of vasoconstriction has tended to overshadow the
contributions of other pathways
(16, 17) . However, there
is now growing evidence for an involvement of PKC in this process. For
example, the activities of calponin and and caldesmon, both involved in
the cross-bridging of actin filaments, are regulated by PKC
phosphorylation (48).
. Although the primary response was only slightly
attenuated by PKC depletion, consistent with previous studies
(49) showing a close coupling between adrenergic receptors and
PLA
in CHO cells, the potentiation of arachidonic acid
release due to concomitant stimulation with PYY was clearly secondary
to PKC activation. Delineation of the site of action of PKC in this
process was not an aim of the current study, but it could be at the
PLA
enzyme itself, or upstream of the activation of the
mitogen-activated protein kinase which is now recognized as an
modulator of PLA
activity
(50) . Whatever the precise
mechanism, the potentiation of PLA
activity by PYY is
likely to be relevant since arachidonic acid has been postulated to
play a role in the regulation of vasoconstriction by inhibiting the
enzyme responsible for dephosphorylation of myosin light
chains
(16) . In functional terms this is the same as stimulating
phosphorylation through the action of the Ca
calmodulin-dependent myosin light chain kinase, and it results in
activation of the myosin Mg
ATPase and contraction of
actin filaments
(16, 17) . The net effect is to render
the contractile apparatus more sensitive to a given rise in
[Ca
]
. Potentiation of
arachidonic acid production would thus be an efficient (but not the
sole) means of linking synergistic increases in PKC activity to
vasoconstriction. An involvement of Y1-NPY receptors in the activation
of PLA
has only been inferred previously from a study
measuring prostaglandin synthesis in vascular endothelial
cells
(51) . The underlying processes were not investigated.
However, it has been demonstrated that other receptor species coupling
to PT-sensitive G proteins are capable of potentiating the arachidonic
acid release initiated by a primary stimulus in CHO cells, without
exerting any stimulation by themselves (52). As was the case with PYY,
the synergistic responses were secondary to activation of
PKC
(52) .
-adrenergic
receptors in order to delineate possible mechanisms underlying the
potentiation of vasoconstriction seen when these receptors are both
occupied in vivo. We have demonstrated a potentiation of PLC
activity using this model, but suggest that the physiological
consequences of this are not evenly distributed between the downstream
pathways of Ca
mobilization and PKC activation. Thus
a synergistic response was seen with PKC but not at the level of
Ca
mobilization. Furthermore, the synergistic
activation of PLA
, an event closely linked to regulation of
the contractile apparatus, was absolutely dependent on the presence of
PKC. We conclude that potentiation of PKC activity is important for the
synergistic responses occurring downstream of the occupation of Y1-NPY
and
-adrenergic receptors.
Table:
Y1-NPY and -adrenergic
receptor-induced inositol phosphate accumulation
, Ins(1,3,4)P
, and
Ins(1,4,5)P
,
respectively.
, phospholipase A
; TPA,
12-O-tetradecanoylphorbol-13-acetate; CHO, Chinese hamster
ovary; tBuHQ, 2,5-di-(t-butyl)-1,4-benzohydroquinone;
PtdInsP
, phosphatidylinositol 4,5-bisphosphate;
Ins(1,4,5)P
, inositol 1,4,5-trisphosphate;
Ins(1,3,4)P
, inositol 1,3,4-trisphosphate;
Ins(1,4)P
, inositol 1,4-bisphosphate;
[Ca
], cytoplasmic free Ca
concentration; PBS, phosphate-buffered saline; KRB, Krebs-Ringer
bicarbonate; MOPS, 3-[N-morpholino)propanesulfonic acid;
PKC, protein kinase C; HPLC, high performance liquid chromatography;
PT, pertussis toxin.
-adrenergic receptor cDNA, and Joe Lynch for
useful comments on the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.