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Vol. 284, Issue 2, 625-632, February 1998
Department of Pharmacological and Physiological Sciences (L.S., R.J.M.) and Medicine (L.H.P.), University of Chicago, Chicago, Illinois
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Abstract |
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Abstract
Introduction
Methods
Results
Discussion
References
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We examined the ability of rat Y1, Y2 and Y4 neuropeptide Y (NPY) receptors to regulate K+ and Ca++ channels expressed in Xenopus oocytes and HEK 293 cells, respectively. Stimulation of all three receptors with NPY or related peptides activated inwardly rectifying K+ currents resulting from the expression of rat GIRK1/CIR in frog oocytes. These effects were inhibited by pertussis toxin treatment. The effects of activating Y1 receptors were antagonized competitively by BIBP3226, SR120819A and GW1229. The effects of Y2 receptor activation were not blocked by these drugs, and the effects of Y4 receptor activation were only blocked by GW1229. Activation of all three NPY receptors also inhibited human alpha-1B Ca++ channels stably expressed in HEK293 cells. The effects of agonists at all three receptors were blocked by pertussis toxin treatment and were voltage dependent. Activation of all three types of NPY receptors produced much smaller inhibition of human alpha-1E Ca++ channels also stably expressed in HEK293 cells. These results suggest that NPY receptors can regulate K+ and Ca++ channels and that these effects may be responsible for the observed effects of NPY on neuronal excitability and synaptic transmission.
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Introduction |
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Abstract
Introduction
Methods
Results
Discussion
References
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Neuropeptide
Y is a 36-amino-acid peptide that is distributed in the peripheral and
central nervous systems (Colmers and Wahlestedt, 1993
). NPY is related
to two other peptides: PYY, which is found mostly in endocrine cells in
the gut, and PP, which is localized mostly to a specific subset of
cells in the endocrine pancreas (Colmers and Wahlestedt, 1993;
Larhammer, 1996). Many investigations have demonstrated the numerous
effects that result from administration of these peptides peripherally
or directly into the brain. These include a host of endocrine actions
(Kalra and Kalra, 1996), as well as central effects on blood pressure
(Grundemar et al., 1991), the dark/light cycle (Huhman and
Albers, 1994), hippocampal excitability (Erickson et al.,
1996) and food consumption (Miller and Bell, 1996). NPY is one of the
most powerful hyperphagic agents known and it is likely that it plays a
central role in the normal regulation of eating behavior (reviewed in
Miller and Bell, 1996). The ability of NPY to increase eating is
thought to result from its effects on neurons in certain parts of the
hypothalamus, including the arcuate and paraventricular nuclei (Kalra
and Kalra, 1996; Stanley, 1993). We recently demonstrated that NPY
could produce presynaptic inhibition of evoked glutamate and
-aminobutyric acid release in the arcuate nucleus and could also
activate a K+ current postsynaptically in a
subpopulation of arcuate neurons (Rhim et al., 1997; Glaum
et al., 1996). Indeed, the ability of NPY to produce
presynaptic inhibition is a common feature of its actions throughout
the central and peripheral nervous systems (Colmers and Bleakman,
1994). We have postulated that this action may be caused by the ability
of NPY to inhibit those neuronal Ca++ channels
that are closely linked to the release of neurotransmitters (Rhim
et al., 1997; Toth et al., 1993). In contrast to
the widely reported ability of NPY to produce presynaptic inhibition,
NPY-induced activation of K+ currents, a feature
commonly associated with the actions of G-protein-linked receptors on
neurons, has only been reported to occur in the arcuate nucleus of rat
brain (Rhim et al., 1997) and in bullfrog sympathetic neurons (Zidichouski et al., 1990).
NPY-related peptides are thought to produce their effects through the
activation of a family of related G-protein-linked receptors. To date
there seem to be five or six members of this family, depending on the
species in question. The Y1, Y2 and Y4 receptors have been studied the
most widely at this point (Herzog et al., 1992; Gerald et al., 1995; Bard et al., 1995). The existence
of a Y3 receptor has been postulated based on the effects of NPY and
related peptides in certain tissues (Grundemar et al.,
1991), but it has never been positively identified by biochemical or
molecular biological criteria. A recently identified Y5 receptor may be
particularly important in the regulation of the feeding response
(Gerald et al., 1996). Finally, a murine "Y5"(Y6)
receptor may exist as a pseudogene in other species (Gregor et
al., 1996). In the present report we describe the effects of the
activation of Y1, Y2 and Y4 receptors on K+ and
Ca++ currents to understand the molecular basis
of the effects of NPY on synaptic communication.
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Methods |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Preparation of cRNA. Complementary DNAs (in the pBluescript II vector, Stratagene, La Jolla, CA) encoding the rat G protein-activated inwardly rectifying K+ channel subunits GIRK1 (Kir 3.1, GenBank L25264) and CIR (Kir 4.1, GenBank L35771), rat neuropeptide receptor Y1 (GenBank Z11504), Y2 and Y4 (GenBank Z68180) were linearized with XbaI, XhoI, ClaI, BamHI and HindIII, and cRNAs were synthesized with T3 polymerase for GIRK1, CIR, Y1 and Y4, and T7 polymerase for Y2 (mMESSAGE mMACHINE, Ambion, Austin, TX).
Expression of cRNAs and recording in Xenopus
oocytes.
Xenopus oocyte injection and recording methods
were as described previously (Philipson et al., 1991; Ma
et al., 1995). Defolliculated oocytes were incubated in a
Petri dish containing OR2 solution (5 mM HEPES, 82.5 mM NaCl, 2.5 mM
KCl, 1 mM MgCl2, 1 mM
Na2HPO4, 1 mM
CaCl2 and gentamicin [0.02 g/l], pH to 7.6 with
NaOH) with a 1% agar layer at the bottom overnight before injection.
; Velimirovic et al., 1996).
RNA aliquots (50 nl) which contained about 20 to 30 ng of each
individual RNA were injected into oocytes. For controls, 50 nl of
diethyl pyrocarbonate (DEPC)-treated water was injected. The injected
oocytes were incubated at 18°C for 60 to 72 hr before recording.
Recording of GIRK1/CIR K+ currents was conducted
with the two-electrode, whole-cell, voltage-clamp technique (Philipson
et al., 1991; Ma et al., 1995). The recording
protocol involved seven pulses stepping from 50 mV to 
130 mV with
30-mV decrements (pulse duration, 300 msec; pulse interval, 8 sec, with
50 msec before and after the train). The oocytes were recorded during
perfusion of OR2 solution, followed by a 50 mM K+
solution (5 mM HEPES, 35 mM NaCl, 50 mM KCl, 1 mM
MgCl2, 1 mM Na2HPO4 and 1 mM
CaCl2, pH to 7.6 with NaOH) with or without
drugs. The effects of NPY analogs (NPY (human), PYY (human),
[Leu31Pro34]NPY (human),
NPY 13-36 (porcine), hPP (human), rPP (rat) (from Sigma, St. Louis,
MO), NPY 2-36 (human, rat) and
[D-Trp32]NPY (human, rat) (from
Bachem, Torrance, CA) on the regulation of K+
currents by NPY receptors were examined. Dose-response curves were
constructed with drugs at (in nM): 0.01, 0.1, 1, 10, 100, 500 and 1000 (in some cases). The data were normalized and analyzed by nonlinear
regression. Four experimental antagonist compounds, BIBP 3226, BIBP
3435 (an inactive isomer of BIBP 3226), SR 120819A and GW1229, were
also tested. For PTX studies, oocytes were incubated with 1.5 to 2 µg/ml PTX overnight before recording.
HEK 293 cell lines and transfection procedures.
The Y1, Y2,
Y4, 
-galactosidase and CD8 cDNAs were subcloned into the mammalian
expression vector pCMV5 (Andersson et al., 1989).
Large-scale plasmid purification for transfections were done with the
Wizard plus Maxipreps-DNA Purification System (Promega, Madison, WI),
Plasmid Maxi Kit (Qiagen, Santa Clarita, CA) or CsCl gradient. HEK 293 G1A1 cells (stably expressing
1B-12B
/1B Ca++ channel subunits) were used to study N-type
Ca++ channels (22) and the HEK 293 E52 cell line
(1E-32B/1B
Ca++ channel subunits) was used to study the
"R" type Ca++ channels (22). HEK 293 cell
lines were grown in Dulbecco's modified Eagle's medium (Gibco, Grand
Island, NY) with geneticin (500 µg/ml) (Gibco), penicillin (100 µg/ml, Gibco), streptomycin (50 µg/ml, Gibco) and 5% fetal calf
serum (HyClone, Logan, UT). One day before the transfection, cells were
plated on poly-L-lysine-coated coverslips. Cells were
cotransfected with different NPY receptor cDNAs and either
cDNA-encoding -galactosidase or CD8. A Ca++
phosphate precipitation method was used for transfection (Toth et
al., 1996). Cells were recorded from 48 to 72 hr after
transfection. Before recording cells were incubated with CD8 (T
cytotoxic/suppressor) Dynabeads (1.5 µl/l, Dynal) for 15 min. CD8
Dynabeads can be seen under the microscope as orange-colored beads.
Cells with beads attached were considered transfected for recording
purposes (Jurman et al., 1994). Receptor synthesis was also
assessed with receptor binding assays, Northern blot analysis and
polymerase chain reaction to confirm the success of the transfection
procedure (data not shown).
Recording of Ca++ currents in transfected
HEK 293 cells.
The recording method was basically as described
previously (Toth et al., 1996). The
Ca++ currents were recorded with use of the
whole-cell patch-clamp technique. Data were acquired with a Axopatch 1D
(Axon Instrument) amplifier, filtered at 2 kHz and stored in the
computer.
,N,-tetraacetic acid (BAPTA) based (100 nM CsCl, 1 mM
MgCl2, 10 mM HEPES, 10 mM BAPTA, 20U/ml creatine
phosphokinase, 5 mM phosphocreatinin, 2 mM MgATP, and 1 mM Tris-GTP).
The cell was first perfused in 2Na-Ca solution (2 mM
CaCl2, 138 mM NaCl, 1 mM
MgCl2, 5 mM KCl, 10 mM HEPES, 10 mM glucose, pH
to 7.4 with NaOH, and osmolarity 300-305), then in
5Ca++-TEA solution (5 mM
CaCl2, 144 mM TEACl, 1 mM
MgCl2, 10 mM HEPES, 10 mM glucose, pH to 7.4 by
TEAOH, and osmolarity 300-305) with and without NPY analogs. Different
NPY analogs and antagonists (as those used in the
K+ study) in 5Ca++-TEA were
tested on Y1, Y2 and Y4 receptors. Dose response curves were also
measured (see above). For PTX studies, cells were treated with 600 ng/ml PTX overnight.
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Results |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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K+ channels in frog oocytes.
We
expressed rat Y1, Y2 and Y4 receptors in frog oocytes together with
K+ channels formed from the combination of the
two subunits GIRK1/CIR (GIRK4) (Krapivinsky et al., 1995).
Addition of NPY or a related peptide produced activation of inwardly
rectifying K+ currents in these oocytes (fig.
1). In contrast, addition of NPY-like
peptides to oocytes that had been injected with water (as a control),
GIRK1 + CIR only, and receptor cRNA only, did not result in activation
of a K+ current (table
1). The ability of all three receptors to
activate K+ currents was inhibited when oocytes
had been incubated overnight with PTX.
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; Matos et
al., 1996; Stanley et al., 1992). NPY(2-36) activated both Y1 and Y2 receptors, but was ineffective at Y4 receptors. [D-Trp32]NPY was relatively
ineffective at all three receptor types.
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), SR120819A
(Serradeil-Le Gal et al., 1995) and GW1229 (Bitran et
al., 1997) all inhibited activation of Y1 receptors in a
competitive fashion. In contrast, the optical isomer of BIBP3226,
BIBP3435, which has been ineffective as an NPY antagonist (Doods
et al., 1995), was ineffective at all three receptors. None
of the antagonists blocked activation of the Y2 receptor. GW1229 also
blocked activation of the Y4 receptor (fig.
2) in a competitive manner, although the
other two antagonists were ineffective (table
3).
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Ca++ channels in the HEK 293 cells.
We
examined the effect of expression of the three NPY receptors on
Ca++ channels stably expressed in HEK 293 cells.
G1A1 cells stably expressed the subunits
1
/1-B/2/
and produced currents that had the properties of N-type
Ca++ currents, whereas E52 cells expressed the
subunits
1E/1-B/2/ and exhibited currents that resembled neuronal "R" currents (Toth et al., 1996). After expression of any of the three NPY
receptors, addition of NPY or a related peptide agonist produced robust
inhibition of Ca++ currents in G1A1 cells (fig.
3A). NPY-like peptides had no effect on
Ca++ currents in cells that were not transfected
with one of the NPY receptors (table 4).
Overnight treatment of cells with PTX abolished the effects of NPY
analogs (table 4).
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;
Hille, 1994). Both steady-state inhibition and slowing of current
activation was observed. Furthermore, the inhibition was voltage
dependent and was relieved to a considerable extent by a depolarizing
prepulse (fig. 4, table
6).
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). This was
also the case in the present series of experiments. Nevertheless, and
unlike our previous studies with opioid and somatostatin (SRIF) receptors (Toth et al., 1996), a degree of inhibition of
alpha-1E Ca++ currents was apparent in
E52 cells (fig. 3B), although these effects were much smaller in
magnitude than those produced in G1A1 cells. We observed such effects
with all three types of NPY receptors. The effects were qualitatively
similar to those seen in G1A1 cells. Slowing of current activation (in
some cases) and steady-state inhibition were observed and the
inhibition was partially relieved with a depolarizing prepulse (fig. 4,
table 6). Inhibition was also blocked by treatment of cells overnight
with PTX (data not shown).
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Discussion |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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The results presented in this paper provide a potential molecular
basis for the reported effects of NPY on synaptic transmission. A
survey of the literature reveals that two major effects of NPY have
been reported. The first of these is presynaptic inhibition of the type
observed at many peripheral neuroeffector junctions, as well as some
central synapses (Rhim et al., 1997; Toth et al., 1993; Obrietan and van den Pol, 1996). A second effect, that has been
described recently, is the ability of NPY to activate a
K+ current in some neurons of the arcuate
nucleus. A similar effect may also occur in bullfrog sympathetic
neurons (Zidichouski et al., 1990).
How are these effects of NPY produced? Although the activation of
K+ currents in neurons by NPY and related
peptides has not been widely reported, all of the three types of NPY
receptors investigated in the present experiments were able to produce
such effects. It is likely that the K+ currents
that are usually activated in neurons as a result of stimulation of
G-protein-linked receptors are members of the GIRK/CIR family, as are
the K+ channels used in the present
investigations (Signori et al., 1997; Rimland et
al., 1996; Brown et al., 1995). In the arcuate nucleus,
only stimulation of Y1 receptors resulted in the activation of
K+ currents of this type (Rhim et al.,
1997). However, it is also likely that these same arcuate neurons
express a variety of NPY receptors (Gerald et al., 1995,
1996; Bard et al., 1995; Gehlert and Gackenheimer, 1997).
Indeed, activation of several types of NPY receptors can inhibit
Ca++ currents in these cells (L. Sun and R. J. Miller, unpublished observations). The receptor activation of
GIRK-like K+ currents and inhibition of
Ca++ currents are both thought to be mediated by
of G-protein beta/gamma subunits (Huang et
al., 1995; Ikeda, 1996). Given that G-protein activation will
occur upon activation of all types of NPY receptors, the relative
selectivity of effects seen in cells such as arcuate neurons suggests
some level of regulation not observed in heterologous expression
systems such as those used here, at least not under our experimental
conditions (e.g., see Schreibmayer et al., 1996).
The ability of NPY to inhibit the evoked release of neurotransmitters
potentially could result from several types of effects that have been
suggested as contributing to presynaptic inhibition (Ikeda, 1996). For
example, first, it has been proposed that activation of a
K+ conductance presynaptically could result in
shunting of the action potential and reduced release (Scholz and
Miller, 1992; Miller, 1990). Second, direct inhibition of those
Ca++ channels in nerve terminals that have been
linked to transmitter release would also be expected to reduce release
(Silinsky, 1985; Scholz and Miller, 1992; Miller, 1990). Finally, it
has been suggested that activation of some G-protein-linked receptors
may produce "direct" effects on the release of neurotransmitters
exerted at some point subsequent to Ca++ entry
(Scholz and Miller, 1992). In studies carried out on sympathetic neuroeffector junctions in culture, we previously demonstrated that
activation of NPY receptors could inhibit Ca++
entry into nerve terminals and suggested that such effects were mainly
responsible for presynaptic inhibition in this case (Toth et
al., 1993). The results reported in this paper are consistent with
this hypothesis. It is clear that activation of all three NPY receptors
tested can inhibit N-type Ca++ channels, one of
the types of Ca++ channels most frequently linked
to transmitter release (Hirning et al., 1988). Analogous
findings have been reported in certain neuronal preparations.
Activation of both Y1 and Y2 NPY receptors have inhibited
Ca++ currents in several types of central and
peripheral neurons (McQuiston et al., 1996; Chen and van den
Pol, 1996; Foucart et al., 1993; Ewald et al.,
1988), and activation of PP receptors (presumably Y4) has resulted in
inhibition of Ca++ currents in a population of
sympathetic neurons (Wollmuth et al., 1995). In this latter
case activation of Y4 receptors produced both rapid regulation of
N-type Ca++ channels and also a second type of
inhibition that seems to proceed by a route that may involve a second
messenger molecule of some type.
As discussed above, recent work has revealed that "rapid, membrane
delimited" receptor regulation of N- and P/Q-type
Ca++ channels is mediated by G-protein
beta/gamma subunits, as is receptor regulation of
GIRK K+ currents (Huang et al., 1995;
Ikeda, 1996). The G-proteins involved in coupling NPY receptors to
Ca++ channels in normal neurons are PTX-sensitive
(e.g., Gi or
Go), which are also present in Xenopus
oocytes (Olate et al., 1989, 1990) and HEK 293 cell lines
(Toth et al., 1996). We and others have recently reported
that Ca++ currents resulting from the expression
of alpha-1E subunits (possibly related to neuronal R-type
Ca++ currents) are much less susceptible to
G-protein-mediated inhibition than Ca++ currents
resulting from the expression of the highly homologous alpha-1B (N type) and alpha-1A (P/Q type)
subunits (Toth et al., 1996; Bourinet et al.,
1996; Page et al., 1997). Indeed, in a previous study we
observed no significant modulation of alpha-1E channels
produced by activation of kappa opioid and somatostatin receptors (Toth et al., 1996). In the present series of
experiments, we actually were able to observe regulation of
alpha-1E currents by NPY receptors, although these effects
were certainly smaller in magnitude than those observed with
alpha-1B. The ability of receptors to regulate
alpha-1E based Ca++ channels is
consistent with the presence of a binding motif for beta/gamma subunits in the second cytoplasmic
loop of alpha-1E (Williams et al., 1994). This is
the site at which beta/gamma subunits may exert
their inhibitory effects on Ca++ channels (De
Waard et al., 1997, but see Zhang et al., 1996). It is also consistent with our observations that
beta/gamma subunits and GTP--S can produce
some modulation of these channels in the same expression system as used
here (Toth et al., 1996; Shekter et al., 1997).
Why the magnitude of G-protein-mediated inhibition of
alpha-1E channels is so much smaller than observed with
alpha-1B and alpha-1A is not yet clear, but may
involve differential interactions with Ca++
channel beta subunits (De Waard et al., 1997). It
is interesting to note that the inhibition of alpha-1E
currents we observed exhibited features that were similar to those
observed for alpha-1B and alpha-1A based
currents. For example, clear kinetic slowing of currents and voltage
dependence of inhibition was apparent. Thus, it is conceivable that
regulation of alpha-1E based Ca++
currents by NPY receptors also occurs in authentic neurons.
The pharmacological selectivity of NPY analogs observed in the present
studies on both K+ and Ca++
currents is generally similar to that already reported in the literature (e.g., Gerald et al., 1996) with
certain exceptions. One difference concerns the activity of NPY(2-36)
which was found to be relatively inactive at Y4 receptors, although in
another recent report it was shown to have reasonable affinity for Y4 receptors by a binding assay (Gehlert et al., 1996). It is
possible, of course, that the differences in some way reflect the use
of heterologous expression systems in the present experiments. A novel
observation we have made concerns the NPY receptor antagonist GW1229.
Although both SR120819A and BIBP3226 proved to be highly selective
antagonists at Y1 receptors, GW1229 also proved to be a potent
antagonist at Y4 receptors and may therefore be useful in this regard.
In summary, the results reported here may provide a basis for some of
the reported effects of NPY on neurons. However, it is clear that
further explanation will be required for some of the phenomena. For
example, the ability of NPY to inhibit evoked glutamate release in
several hypothalamic nuclei is very long lasting and is apparent for
many minutes after washout of the agonist (Rhim et al.,
1997; Obrietan and van den Pol, 1996). On the other hand, the effects
of NPY on K+ and Ca++
currents reported here reverse quite rapidly. Why this difference occurs remains to be explained.
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Acknowledgments |
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We are indepted to SIBIA Neurosciences and Eli Lilly for supplying the G1A1 and E52 cell lines, to Synaptic Pharmaceuticals for supplying Y1, Y2 and Y4 receptor clones, to Don Gehlert (Eli Lilly Co.) for optical isomers of BIBP, to Eric Parker (Schering-Plough) for SR120819A and to Alejandro Daniels (Glaxo-Wellcome) for GW1229. We thank Dong-Jun Ren for technical assistance.
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Footnotes |
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Accepted for publication October 30, 1997.
Received for publication July 21, 1997.
1 Supported by PHS grants DA-02121, MH-40165, DA-02575, DK-42086, DK-44840 and NS-33502 from the National Institutes of Health.
Send reprint requests to: Richard J. Miller, Ph.D., Department of Pharmacological and Physiological Sciences, The University of Chicago, 947 E. 58th Street (MC 0926), Chicago, IL 60637.
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Abbreviations |
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NPY, neuropeptide Y;
PYY, peptide YY;
PP, pancreatic polypeptide;
PTX, pertussis toxin;
HEPES, N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid;
TEA, tetraethylammonium.
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References |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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i1 subunits in activation of G-protein gated inward rectifying K+ channels (GIRK1) by human NPY1 receptors.
Br J Pharmacol
116:
2346-2348[Medline]. / complex to voltage dependent calcium channels.
Nature
385:
442-446[Medline]. / to the GIRK1 G-protein gated inwardly rectifying K+ channel is important for channel activation.
Neuron
15:
1133-1143[Medline]. / subunits.
Nature
380:
255-258[Medline]. 
-Opioid receptor activates an inwardly rectifying K+ channel by a G protein-linked mechanism: Coexpression in Xenopus oocytes.
Mol Pharmacol
47:
1035-1040[Abstract]. -subunit of a G0-type protein of Xenopus laevis oocytes.
FEBS Lett
244:
188-192[Medline]. -subunits of G proteins from Xenopus laevis oocytes.
FEBS Lett
268:
27-31[Medline]. -subunits.
Nature
380:
624-627[Medline]. / subunits.
Mol Pharmacol
52:
282-2911E calcium channel subtypes.
J Biol Chem
269:
22347-22357
0022-3565/98/2842-0625$03.00/0
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1998 by The American Society for Pharmacology and Experimental Therapeutics
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