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The Journal of Neurophysiology Vol. 85 No. 5 May 2001, pp. 1888-1898
Copyright ©2001 by the American Physiological Society
Department of Anatomy and Histology and Centre for Neuroscience, Flinders University of South Australia, Adelaide, SA 5001, Australia
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Jobling, Phillip, Jennifer P. Messenger, and Ian L. Gibbins. Differential Expression of Functionally Identified and Immunohistochemically Identified NK1 Receptors on Sympathetic Neurons. J. Neurophysiol. 85: 1888-1898, 2001. We have used multiple-labeling immunohistochemistry, intracellular dye-filling, and intracellular microelectrode recordings to characterize the distribution of tachykinin receptors and substance P boutons on subpopulations of neurons within the guinea pig celiac ganglion. Superfusion of substance P (SP, 1 µM for 1 min) depolarized 42% of tonic neurons and inhibited afterhyperpolarizations in 66% of long afterhyperpolarizing (LAH) neurons without significant desensitization. Twenty-one percent of tonic neurons and 24% of LAH neurons responded to the NK3 agonist senktide but did not respond to SP, indicating SP did not activate NK3 receptors at this concentration. All effects of SP were abolished by the selective NK1 receptor antagonist, SR140333, but not by the selective NK3 receptor antagonist, SR142801, suggesting that exogenous SP activated a receptor with NK1 pharmacology. No dye-filled LAH neuron and only 50% of tonic neurons responding to SP expressed NK1 receptor immunoreactivity (NK1-IR). All neurons responding to SP had SP immunoreactive fibers within one cell diameter, indicating good spatial matching between SP release sites and target neurons. These results indicate that SP may act via a receptor with NK1-like pharmacology that has a C terminus not recognized by antibodies to the intracellular domain of the conventional NK1 receptor. Inward currents evoked by SP acting on this NK1-like receptor or senktide acting through NK3 receptors had identical current-voltage relationships. In LAH neurons, both agonists suppressed IsAHP without reducing IAHP. Responses evoked by SP and senktide were resistant to PKC inhibitors, suggesting that the transduction mechanisms for the NK1-like receptor and the NK3 receptor may be similar.
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Within the nervous system of
mammals, tachykinins such as substance P (SP) are thought to act via
three well-defined subclasses of neurokinin (NK) receptor:
NK1, NK2, and
NK3 receptors (Iversen 1994
;
Krause et al. 1994). Classification of NK receptors was based originally on the relative potency of several endogenous and
synthetic tachykinin agonists (Regoli et al. 1994).
Subsequent development of specific antagonists and the eventual cloning
of three main subtypes of rat and human NK receptor have supported the
concept of a restricted number of receptors (Krause et al. 1994). Although evidence for a short form of the
NK1 receptor has recently surfaced, physiological
roles or distributions of this variant within the nervous system have
yet to be determined (Fong et al. 1992)
Participation of tachykinins in synaptic transmission has been found
throughout the nervous system and is present in a wide variety of
species. It has been particularly well characterized in the enteric
nervous system (Holzer and Holzer-Petsche 1997; Johnson et al. 1998), and spinal cord (Parker and
Grillner 1996; Urban et al. 1994). Within guinea
pig prevertebral ganglia, SP is contained in axon collaterals of
sensory neurons (Dalsgaard et al. 1983;
Kreulen and Peters 1986; Mathews and Cuello 1982, 1984) as well as a subpopulation of preganglionic boutons
(Gibbins 1995). Evidence for the involvement of
tachykinins in synaptic transmission within the celiac ganglion was
first obtained two decades ago (Konishi et al. 1983;
Tsunoo et al. 1982). More recently, Zhao and colleagues
(Zhao et al. 1995, 1996) have elucidated the pharmacological profile of the tachykinin receptors responsible for the
effects of both exogenously and endogenously released SP on different
electrophysiological classes of neurons within this ganglion.
Stimulation of the celiac nerves evoked a depolarization in
electrophysiologically defined tonic neurons and inhibition of the
afterhyperpolarization in long afterhyperpolarizing (LAH) neurons. Both
responses were inhibited by the NK1 antagonist
GR71251 (Zhao et al. 1996). Furthermore, responses to
exogenous SP and neurokinin A (NKA) were also blocked by GR71251,
indicating that SP acts via NK1 receptors in this
ganglion and that NK2 receptors are absent.
We have recently investigated the anatomical distribution of both SP
binding sites and NK1 receptor immunoreactivity
within subpopulations of neurons in the guinea pig celiac ganglion
(Messenger and Gibbins 1998; Messenger et al.
1999). In this ganglion, subpopulations of neurons are
topographically separated. LAH neurons contain NPY and are
preferentially located in the lateral regions of the ganglion, whereas
tonic neurons are located medially and contain either somatostatin or
no known peptide (Costa and Furness 1984; Gibbins
et al. 1999; Keast et al. 1993; Macrae et
al. 1986). We found that both SP binding and immunoreactivity
to NK1 receptors (NK1-IR)
occur preferentially on neurons that lack NPY and occur in the medial
celiac ganglion; these are likely to be tonic neurons projecting to the
gut. In contrast, very few NPY-containing neurons, which occur in the
lateral celiac ganglion and project mostly to blood vessels, showed
NK1-IR (Messenger et al. 1999).
This differential distribution of immunohistochemically identified NK1 receptors is in contrast with the
pharmacological studies of Zhao et al. (1995, 1996),
which suggest that NK1 receptors are found on a
majority of both LAH and tonic neurons. To resolve this issue, it is
necessary to determine the distribution of functionally and
immunohistochemically identified NK1 receptors on
a cell-by-cell basis. Therefore we have used a combined
pharmacological, electrophysiological, and immunohistochemical approach
to investigate the distribution of NK1 receptors
on individual LAH and tonic neurons within the celiac ganglion. As we
previously identified a spatial mismatch between
NK1-IR neurons and SP-IR boutons
(Messenger et al. 1999), we also compared the
relationship between neurons which respond to exogenous SP and the
distribution of SP-IR nerve fibers.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Male or female guinea pigs weighing 160-350 g were used. The celiac ganglion was dissected from guinea pigs that had been stunned by a blow to the head and bled via the carotid arteries. These procedures were approved by the Animal Welfare Committee of the Flinders University of South Australia.
Intracellular electrophysiology
Neurons in the celiac ganglion were prepared for intracellular
electrophysiological recordings as described previously (Gibbins et al. 1999; Jobling and Gibbins 1999). Briefly,
celiac ganglia were isolated and placed in a HEPES-buffered balanced
salt solution [composition was (in mM) 146 NaCl, 4.7 KCl, 0.6 MgSO4, 1.6 NaHCO3, 0.13 NaH2PO4, 2.5 CaCl2, 7.8 glucose, and 20 HEPES, buffered to pH
7.3) bubbled with oxygen. Ganglia were pinned to the base of a
recording chamber (1 ml) coated with silicone elastomer (Sylgard, Dow
Corning, Midland MI), maintained at 35°C, and perfused with physiological solution at 2.5 ml/min. Neurons were impaled with glass
microelectrodes filled with 0.5 M KCl, having resistances of 80-200
M
. In some cases, 0.5% wt/vol Neurobiotin (Vector, Burlingame, CA)
was included in the electrode filling solution to enable postimpalement
visualization of neurons.
Data were recorded using an Axoprobe 1A or an Axoclamp 2B amplifier
(Axon Instruments, Burlingame, CA). Voltage and current records were
digitized at 1-5 kHz using with either a 1401-Plus A/D converter
running Spike2 software (version 3.0, Cambridge Electronic Design,
Cambridge, UK) or a Maclab running Chart and Scope software (version
3.6, ADI Instruments, Castle Hill, NSW, Australia). Digitized data were
analyzed using Igor Pro (version 3.14, WaveMetrics, Lake Oswego,
OR). Changes in membrane potential were determined using either
discontinuous current clamp (DCC) or bridge mode. Single-electrode
voltage clamp (SEVC) was used to measure currents underlying the AHP.
During DCC and SEVC, the headstage was continuously monitored and the
cycling frequency adjusted to minimize the effects of electrode
capacitance. The cycling frequency was 1.0-2.0 kHz for DCC and 2-3.5
kHz for SEVC. The tail currents underlying the AHP were measured after
suprathreshold voltage steps (10 ms) initiated a single brief "action
current" corresponding to an unclamped action potential
(Jobling and Gibbins 1999; Jobling et al.
1993; Wang and McKinnon 1995). Neurons were classified as tonic on the basis of their continuous action potential discharge throughout a depolarizing current injection (250-ms duration). LAH neurons were classified on the basis of a prolonged afterhyperpolarization (>1 s) following a single action potential evoked by a 10- to 50-ms depolarizing current injection (see
Keast et al. 1993; Weems and Szurszewski
1978).
Drugs used
SP and senktide were obtained from Auspep (Parkville, VIC,
Australia). Both agonists were dissolved in distilled
H2O at a concentration of 1 mM and stored frozen
in 10-µl aliquots. SP and senktide were made up to their final
concentrations in HEPES-buffered salt solution immediately prior to
use. They were applied to the preparation by switching the perfusion
lines between normal and agonist containing solution for 1 min. SP was
routinely used at a concentration of 1 µM and senktide at 0.5 µM as
preliminary observations, and published data for these (Zhao et
al. 1995) and other peripheral neurons (Hardwick et al.
1997) indicated that these concentrations were close to
maximal. SP (1 µM) or senktide (0.5 µM) could be applied to the
preparation every 10 min without any evidence of desensitization (Fig.
1). In most experiments, senktide and SP
were alternately applied at 10-min intervals (Fig. 1). Neurons were
considered to be responsive to tachykinin agonists if the resting
membrane potential depolarized by 
2 mV, which was the smallest shift
that could unambiguously be detected above baseline variance.
Additionally, LAH neurons were considered responsive to agonist if the
AHP decreased by 1 mV 1 s after the action potential. The
NK1 receptor antagonist SR140333 and
NK3 receptor antagonist SR142801 were a generous gift of Sanofi Recherche (Montpellier, France). These were dissolved in
dimethylsulfoxide (DMSO) and stored frozen at a concentration of 10 mM
until use. NK receptor antagonists were used at a final concentration
of 1 µM with all measurements in antagonist-containing solution made
after at least 30 min exposure. Staurosporine,
1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), and
N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA1004) were
obtained from Sigma (Castle Hill, Australia). Bisindolylmaleimide (Bis)
was obtained from Calbiochem (La Jolla, CA).
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Visualization of NK1 receptors
Following electrophysiological recording, ganglia were immersed
in fixative (0.2% picric acid and 2% formaldehyde in 0.1 M phosphate
buffer, pH 7.0) overnight at 4°C. The tissue was cleared of fixative
and dehydrated in a graded series of ethanol, three washes of DMSO, and
finally in 100% ethanol. The ganglia were infiltrated with
polyethylene glycol (PEG, MW 1000; Sigma) at 60°C, under vacuum, for
30 min and then mounted in PEG, MW 1450 (Murphy et al.
1998). Sections 30 µm thick were cut at room temperature, initially placed into phosphate-buffered saline (PBS), and then incubated in 10% normal donkey serum (NDS) for 30 min. The sections were incubated in primary antiserum containing 10% NDS for 48 h.
Immunoreactivity to NK1 receptors was detected
with an antiserum directed toward the intracellular C terminus of the
NK1 receptor. This antiserum does not cross-react
with NK2 or NK3 receptors (Shigemoto et al. 1993). Previous studies in the celiac
ganglion of guinea pigs showed that the distribution of
NK1 receptor immunoreactivity observed with this
antiserum matches the distribution of high-affinity binding sites for
SP at NK1 receptors as determined by high
resolution autoradiography (Messenger and Gibbins 1998;
Messenger et al. 1999). The distribution of this
NK1 receptor immunoreactivity was identical to
that seen with another antiserum that also was raised against the
intracellular portion of the NK1 receptor
(Vigna et al. 1994; see Messenger et al.
1999). Other primary antisera used were raised against
somatostatin (Som), raised in mouse (gift from Dr. J. C. Brown,
clone SOMA 08), or SP, raised in rat (clone NC1/34HL, Sera Lab).
Primary antisera were washed off in PBS, and the tissue was incubated
in species-specific secondary antisera raised in donkeys for 1 h.
Species-specific secondary antisera were conjugated to Cy3, Cy5,
dichlorotriazinylamino fluorescein (DTAF), or
7-amino-4-methylcoumarin-3-acetic acid (AMCA), all obtained from
Jackson Immunoresearch Laboratories (West Grove, PA). For visualization
of Neurobiotin, streptavidin conjugated to AMCA or Cy5 was used.
Previous studies in our laboratory have shown that this dye-filling
procedure and the associated intracellular recording of the electrical
properties of the neurons does not diminish their immunoreactivity for
a range of neuropeptides and enzymes (Gibbins et al.
1999).
Labeled sections were mounted in carbonate-buffered glycerol (pH 8.6), sealed with nail varnish, and examined with an Olympus BX50 epifluorescence microscope, fitted with highly discriminating filters (Chroma Technology). Images were obtained with a Sony monochrome CCD video camera (Model SSC-M370CE) and were captured on a Macintosh PowerPC computer, using NIH Image v1.60b7 software. The images were the average of 16 accumulated frames. Final images were assembled with Adobe Photoshop, version 5.02, adjusting only contrast and brightness. Some images of dye-filled neurons were processed using a blind deconvolution algorithm (AutoDeblur, version 6, AutoQuant Imaging, Watervliet, NY) to maximize the definition of the dendritic arbors.
Statistical analysis
Summary data are presented in the text as means ± SE.
Error bars shown in the figures also represent SE. Percentages are
expressed with 95% confidence limits derived from the binomial
distribution (Rohlf and Sokal 1995). Data were analyzed
using t-tests, repeated-measures ANOVA, or
2 tests using SPSS for Windows (version 9, SPSS, Chicago, IL).
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Recordings were generally made from LAH or tonic neurons as they
account for most of the neurons within the celiac ganglion (Keast et al. 1993; Zhao et al. 1995). As
described previously, LAH neurons were preferentially located in
lateral poles of the ganglion, whereas tonic neurons were located
medially (Gibbins et al. 1999; Keast et al.
1993).
Proportions of neurons responding to SP and senktide
Perfusion of 1 µM SP for 1 min evoked a depolarization in 42%
(95% confidence limits: 20-55%) of tonic neurons. In LAH neurons, the most prominent effect of 1 µM SP was a reduction in the amplitude and duration of the AHP (Fig. 1B), which was accompanied by
a depolarization of variable amplitude. Responses to SP (1 µM) were observed in 66% (95% confidence limits: 51-79%) of LAH neurons; this was significantly more than the proportion of tonic neurons responding to SP (2 = 4.9; df = 1, P < 0.02). The NK3 receptor
agonist senktide (0.5 µM) evoked a depolarization in 51% (95%
confidence limits: 37-65%) of tonic neurons and AHP inhibition in
91% (95% confidence limits: 76-98%) of LAH neurons
(2 = 12.6; df = 1, P < 0.001). In neurons where both agonists were tested, 21% of tonic neurons and 24% of LAH neurons responded to
senktide but showed no response to SP (Fig. 2,
A-C). Forty percent of tonic
neurons failed to respond to either agonist, whereas only 9% of LAH
neurons did not respond to either agonist (2 = 15.7; df = 3, P < 0.002, Fig. 2C).
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Relationship between responses to SP and the presence of NK1 receptor
As described previously, NK1-IR was
distributed preferentially on neurons within the medial celiac
ganglion, where tonic neurons predominate, and no
NK1-IR was observed on cells in the lateral regions of the ganglion containing predominantly LAH neurons (see Messenger et al. 1999). To test the relationship between
the immunohistochemical and pharmacological identification of
NK1 receptors, individual neurons were tested for
a response to 1 µM SP and filled with neurobiotin followed by
labeling for NK1-IR. Fifty percent (6 of 12) of
tonic neurons that responded to SP with a depolarization also expressed
NK1-IR (Figs. 3 and
5Aa). Only 1 of 21 tonic neurons did not respond to SP but
did express NK1-IR. The magnitude of the
depolarization in response to SP was twice as large in tonic neurons
that did express NK1-IR (9.3 ± 2.0 mV)
compared with neurons that did not express detectable
NK1-IR (4.3 ± 0.9 mV, P = 0.04).
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In contrast to tonic neurons, none of nine LAH neurons that responded
to SP with a depolarization and an inhibition of the LAH expressed
NK1-IR (2 = 8.9, df = 3, P = 0.03; Figs. 4
and 5Ab). These proportions of
tonic and LAH neurons that showed NK1-IR after
dye filling were consistent with predictions from our previous
immunohistochemical studies (Messenger et al. 1999).
Furthermore there was no evidence for a significant degree of
internalization of NK1-IR following stimulation
of the neurons with SP (cf. Jenkinson et al. 1999; Southwell et al. 1996).
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Relationship between responses to SP and SP-IR axons
Most tonic neurons were surrounded by varicose SP-IR axons (Figs. 3 and 5Ba). However, only 56% of tonic neurons (95% confidence limits: 37-73%) that had SP-IR axons within one cell diameter responded to 1 µM SP (Figs. 3D and 5Ba). In contrast, most LAH neurons (10 of 12) that had SP-IR axons within one cell diameter responded to SP (Figs. 4C and 5Bb).
Pharmacological analysis of SP- and senktide-evoked responses
Evidence from Zhao and colleagues (1995) suggests
that SP selectively activates NK1 receptors and
senktide selectively activates NK3 receptors
within the guinea pig celiac ganglion. Given the lack of correlation
between the expression of NK1 receptor
immunoreactivity and the responsiveness of neurons to SP, we decided to
use selective NK1 and NK3
receptor antagonists to confirm the receptor selectivity of SP and senktide.
LAH NEURONS. The ability of SP to inhibit the AHP in LAH neurons was antagonized by the selective NK1 receptor antagonist SR140333 (1 µM; Fig. 7Ec). SP reduced the magnitude of the AHP area by 58 ± 12% (n = 4) in control solution (t = 8.1, P = 0.004) but had no effect in the presence of SR140333 (Figs. 6A and 7Ea), indicating that SP acts via NK1 receptors. The reduction of AHP area by SP was not affected by the selective NK3 receptor antagonist SR142801 (n = 3, t = 0.5, P = 0.7, Figs. 6B and 7Eb).
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TONIC NEURONS.
The depolarization evoked by SP in tonic neurons was reduced in
four of five neurons by 79 ± 28% in the presence of 1 µM
SR140333 (t = 6.2, P = 0.008; Fig. 7,
A and Da). The response of the remaining neuron
was not affected by SR140333. In contrast, SR142801 did not inhibit the
SP-evoked depolarization (t = 
2.1, P = 0.1; n = 4, Fig. 7C). The depolarization
evoked by senktide was abolished in the presence of the
NK3 receptor antagonist SR142801 (Fig. 7B).
Effects of SP and senktide on membrane properties
Depolarizations evoked by SP or senktide in both tonic
and LAH neurons were accompanied by an increase in input resistance (current clamp) or decreases in input conductance (voltage clamp; Fig.
8Aa). Voltage ramps (80 mV/s)
between 120 and 40 mV were evoked in the absence and in
the presence of either SP or senktide. Subtraction of the resulting
currents in agonist from those in control solution revealed the voltage
dependence of SP and senktide inward currents (Fig. 8B). In
three of three tonic and two of two LAH neurons, currents evoked by SP
reversed between 72 and 98 mV (mean: 89.0 ± 4.5 mV,
n = 5). Senktide-evoked currents in two tonic and one
LAH neuron reversed between 102 and 110 mV. This observation
suggests that both SP and senktide acted predominantly through
decreases in K+ conductance.
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Two major calcium activated K+ currents underlie
the AHP in LAH neurons, IAHP and
IsAHP (Sah 1996). To
determine which conductances were decreased during AHP inhibition by SP
or senktide, the peak amplitudes of
IAHP and
IsAHP were measured before and in the
presence of these agonists (Fig. 8C). SP significantly
inhibited IsAHP by 62 ± 6%
(n = 12, t = 4.4, P < 0.001) without reducing IAHP (n = 12, t = 0.1, P = 0.9; Fig. 8C, a and c). Senktide also reduced IsAHP (78 ± 6% reduction,
n = 3, t = 4.9, P < 0.02) but not IAHP (n = 4, t = 0.3, P = 0.8; Fig. 8C,
b and d). This observation is consistent with the
results of Zhao et al. (1995), who reported that AHP
reduction occurred selectively in LAH but not tonic neurons. There was
no significant correlation between the magnitude of the reduction in
AHP area by SP (n = 11, R = 0.2, P = 0.2) or senktide (n = 9, R = 0.3, P = 0.2) and the level of
depolarization evoked by these agonists. Similarly, in voltage clamp,
there was no correlation between the SP-evoked inward current and the
SP-evoked reduction in IsAHP
(n = 10, R = 0.01, P = 0.9).
Effects of protein kinase inhibitors on responses to SP and senktide
Many neuronal effects of tachykinins have been linked to
generation of protein kinase C (Bertrand and Galligan
1995; Parker et al. 1997; Takano et al.
1995). To determine whether responses mediated by SP or
senktide were mediated through PKC-dependent pathways, several PKC
inhibitors were used. The PKC inhibitor Bis failed to abolish the AHP
inhibition mediated by SP or senktide. In four LAH neurons, 1 µM SP
reduced AHP area by 59 ± 4% in control solution and 51 ± 13% in the presence of 10 µM Bis. Senktide reduced AHP area in these
neurons by 77 ± 7% in control solution and 80 ± 2% in 10 µM Bis. Similarly H7 (10 µM, n = 2) or HA1004
(n = 2) did not change the inhibition of
IsAHP following SP perfusion. In tonic
neurons, the nonspecific protein kinase inhibitor staurosporine (0.1-0.5 µM) did not alter the depolarization evoked by SP in tonic
neurons (n = 3).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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We have investigated the distribution of NK1 receptors using pharmacological, electrophysiological and immunohistochemical techniques in the guinea pig celiac ganglion. Even though the actions of SP appear to be mediated by receptors with the pharmacology of NK1 receptors, we found little correlation between the expression of immunoreactivity to NK1 receptors and sensitivity to SP in individual neurons.
Pharmacological identification of NK tachykinin receptors
There is pharmacological evidence for the presence of both
the NK1 and NK3 subtypes of
tachykinin receptors, but not the NK2 subtype, on
neurons of the guinea pig celiac ganglion (Messenger and Gibbins
1998; Zhao et al. 1995, 1996). Actions of SP on
tonic and LAH neurons were selectively inhibited by the
NK1 receptor antagonist SR140333, which had no
effect on the actions of senktide, a selective
NK3 agonist. SR140333 is a nonpeptide antagonist
that is highly selective for NK1 receptors
(Emonds-Alt et al. 1993). Furthermore this antagonist
has been shown to be selective against the guinea pig neuronal
NK1 receptor (Nalivaiko et al.
1997). These findings support previous experiments by
Zhao et al. (1995, 1996), who found that responses to SP
were inhibited by the NK1 receptor antagonist GR71251.
Responses to SP were not altered by the selective
NK3 receptor antagonist SR142801. This antagonist
has also been shown to be selective at the guinea pig neuronal
NK3 receptor (Nalivaiko et al.
1997). Further evidence that SP does not activate
NK3 receptors comes from the observation that SP
failed to excite many neurons that were markedly excited by senktide.
Although Zhao et al. (1995, 1996) did not use an
NK3 receptor antagonist in their studies, they
too found neurons that were responsive to senktide but not SP.
Together, these observations suggest that, under the experimental conditions used in this study and that of Zhao et al. (1995,
1996), SP does not normally activate NK3
receptors to any significant degree.
Immunohistochemical localization of NK1 receptors
NK1 receptor immunoreactivity was not found
on any LAH neurons despite the fact that the majority of these neurons
responded to SP. Furthermore, only half the tonic neurons that
responded to SP were NK1-IR. This is consistent
with our previous celiac-ganglion study, which showed that
NK1-IR was absent from most NPY-containing neurons, that is, those neurons likely to be LAH neurons, and NK1-IR was localized to a subset of neurons
without NPY, most of which are likely to be tonic neurons
(Messenger et al. 1999). The results presented here are
also consistent with our previous autoradiographic study showing that
high-affinity binding sites for SP with pharmacological characteristics
of NK1 receptors were restricted to the medial
regions of the celiac ganglion containing predominantly tonic neurons
and were absent from the lateral regions of the ganglion containing
mainly LAH neurons (Messenger and Gibbins 1998). On the
basis of the consistency of these results, there is no reason to think
that prolonged intracellular recording diminished NK1-IR in these neurons (see also Gibbins
et al. 1999).
Mismatch between "pharmacological" and "anatomical" NK1 receptors
These present results explain the apparent anomaly between our
previous immunohistochemical study showing a restricted
NK1 receptor distribution and the pharmacological
studies of Zhao and colleagues (1995) that suggested a
wider distribution of NK1 receptors. There is
clearly a mismatch between the distribution of
NK1 receptors identified pharmacologically versus
those identified with immunohistochemistry. A similar situation may
occur in the submucosal plexus of the guinea pig ileum, where the
majority of neurons responding to NK1 receptor
agonists did not express NK1 receptor
immunoreactivity (Moore et al. 1997). Furthermore, submucous arterioles that dilate in response to SP via
NK1 receptor activation (Moore et al.
1997) do not express NK1-IR
(Portbury et al. 1996). These workers concluded that two
variants of the NK1 receptor were involved and
that the receptor responsible for neuronal depolarization was not
recognized by NK1 receptor antibodies.
Subtypes of NK1 receptors
The "three peptides, three receptors" view of NK receptors has
increasingly come into question (see Maggi and Schwarz
1997). This is particularly apparent with pharmacological
studies of NK1 receptors where the existence of a
"septide-sensitive" NK1-related receptor has
been debated (Glowinski 1995; Jenkinson et al.
1999; Maggi and Schwartz 1997; Petitet et
al. 1992). Moreover, there is evidence for a splice variant of
the NK1 receptor that has a truncated
intracellular carboxy terminus (Fong et al. 1992; Kage et al. 1993). There are several lines of evidence
that a similar form of the NK1 receptor may
transduce the actions of SP on neurons in the celiac ganglion, in
particular on the LAH neurons where no NK1-IR
could be detected. First, because the antisera used in this study are
directed against the intracellular carboxy terminus of the conventional
NK1 receptor (Shigemoto et al.
1993), they would not recognize a variant that was truncated in
this region. Second, unlike the extended form of the receptor (Vigna 1999), the truncated form of the receptor does
not show significant desensitization to repeated applications of
agonists (Li et al. 1997; Sanders and Le Vine
1996) and probably is not internalized after activation
(Böhm et al. 1997). Consistent with this, neurons
in the celiac ganglion responded repeatedly to high concentrations of
SP over long periods of time without any signs of desensitization or
receptor internalization. This observation, together with the lack of
high-affinity SP binding sites on LAH neurons (Messenger and
Gibbins 1998), also is consistent with observations that at
least some forms of the truncated receptor have a decreased affinity
for SP compared with the conventional NK1
receptor (Fong et al. 1992).
The actions of the conventional NK1 receptor
generally are thought to be mediated by G-proteins which are linked to
its intracellular domains (Böhm et al. 1997;
Roush et al. 1999). Some of these G proteins can then
activate a variety of kinases including PKC and PKA. However, we were
unable to block the effects of SP or senktide with a range of protein
kinase inhibitors, which are effective against the actions of
tachykinin agonists in other types of neurons (Bertrand and
Galligan 1995; Parker et al. 1997; Takano
et al. 1995). Once again, these observations are consistent with the presence of a receptor with a truncated intracellular sequence.
Actions of SP and senktide
SP and senktide had identical actions on both tonic and LAH
neurons, suggesting that pharmacologically identified
NK1 and NK3 receptors are
transduced through similar pathways to inhibit the same ionic
conductances. In sympathetic neurons, SP is known to alter many ionic
conductances including suppression of several K+
conductances, activation of a nonspecific cation conductance, and
inhibition of N-type calcium channels (Adams et al.
1983; Gilbert et al. 1998; Jones
1985; Shapiro and Hille 1993; Vanner et
al. 1993). The depolarization evoked by SP and senktide in both
tonic and LAH neurons was associated with a decrease in input resistance with the underlying inward current reversing at around 100
mV. This suggests that suppression of K+
conductances dominates the depolarization evoked by both these agonists
in nondissociated celiac ganglion neurons. Our observation that the
size of the depolarization was greater in tonic neurons that expressed
NK1-IR compared with tonic neurons that lacked NK1-IR raises the further possibility that both
the conventional NK1 receptor and the form not
recognized by the antibody may independently inhibit similar conductances.
The slow AHP in LAH neurons results from activation of a
calcium-activated K+ current
(IsAHP) (Keast et al.
1993; Sah 1996). In central neurons, IsAHP is thought to be due to
activation of a novel channel with slow kinetics (Sah
1996). However, in LAH neurons, the slow AHP may result from
the simultaneous activation of three separate calcium-activated
K+ channels: BK, SK, and the novel slow channel
(Martinez-Pinna et al. 2000). In enteric AH neurons,
BK-type calcium-activated K+ channels contribute
to resting membrane potential (Tokimasa and Akasu 1995),
suggesting that part of the depolarization to SP in those neurons is
due to suppression of this resting current. Although there is some
evidence for a contribution of IsAHP
to resting membrane potential in LAH neurons (Martinez-Pinna et
al. 2000), it is unlikely that suppression of this current
alone accounts for the tachykinin-induced depolarization in these
neurons. There was little correlation between the magnitude of AHP
reduction by SP or senktide and the amount of depolarization evoked by
these agonists. Furthermore in cultured LAH neurons, SP is associated with reductions in M current and an instantaneous leak conductance (Vanner et al. 1993). Without further experiments
involving selective ion channel antagonists, it is difficult to
quantify which channels contribute most to membrane depolarization in
either LAH or tonic neurons. However, muscarinic depolarization in
guinea pig celiac neurons involves suppression of five distinct
K+ conductances, suggesting that activation of a
single G-protein-coupled receptor can have widespread effects in these
neurons (Cassell and McLachlan 1987).
The signal transduction mechanisms utilized by tachykinin receptors in
celiac ganglion neurons are not clear. The lack of effect of a range of
kinase inhibitors tends to rule out the involvement of PKC and PKA in
the tachykinin-mediated suppression of K+
conductances in prevertebral sympathetic neurons. In amphibian paravertebral sympathetic neurons where the major effect of SP is
suppression of M current, PKC antagonists are also ineffective (Bosma and Hille 1989; Nakajima and Nakajima
1994). This is similar to the muscarinic suppression of M
current in both amphibian and mammalian paravertebral sympathetic
neurons where the signal transduction mechanism remains elusive
(Marrion 1997).
Conclusions
The immunohistochemical visualization of NK receptors has been a
widely used tool for studying neurotransmission mediated by
tachykinins. Several studies have reported an apparent mismatch between
the distribution of NK1 receptors and release
sites for SP (Liu et al. 1994; Messenger et al.
1999; Nakaya et al. 1994) and have suggested
that SP may act primarily via volume transmission. However, if there is
a population of NK1-like receptors that are not
seen by the current range of antisera, the occurrence of volume transmission by tachykinins may not be as widespread as predicted by
those results. Indeed in the present study, the good correlation between the presence of functionally identified
NK1 receptors and nearby SP-IR fibers implies
that it is not necessary to evoke the concept of volume transmission
for SP in the celiac ganglion. Further studies will be required to
determine the distribution and functional significance of a truncated
form of tachykinin receptor that seems to be responsible for mediating
actions of tachykinins in mammalian sympathetic neurons.
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ACKNOWLEDGMENTS |
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We thank Dr. Ryuichi Shigemoto (Department of Morphological Brain Science, Faculty of Medicine, Kyoto University, Kyoto, Japan) for the gift of antiserum to NK1 receptor and Dr. J. C. Brown (Medical Research Council of Canada Regulatory Peptide Group, Department of Physiology, University of British Columbia, Vancouver, Canada) for the gift of antiserum to somatostatin. We also thank Assoc. Prof. J. Morris, Dr. S.J.H. Brookes, Dr. V. Zagorodnyuk, and R. L. Anderson for helpful advice on the manuscript.
This study was supported by grants from the National Health and Medical Research Council of Australia, the Charles and Sylvia Viertel Foundation, the Clive and Vera Ramaciotti Foundation, the Flinders Medical Centre Foundation, the Australian Research Council, and the Flinders University Research Budget.
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FOOTNOTES |
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Address for reprint requests: P. Jobling, Dept. of Anatomy and Histology and Centre for Neuroscience, Flinders University of South Australia, GPO Box 2100, Adelaide, SA 5001, Australia (E-mail: phillip.jobling{at}flinders.edu.au).
Received 1 September 2000; accepted in final form 4 January 2001.
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ABSTRACT
INTRODUCTION
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DISCUSSION
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) was immunoreactive for both
NK1 receptor (B) and Somatostatin
(C). D: some SP-IR fibers lie close to this
neuron. E: depolarization evoked by 1 µM SP in this
neuron.
, relative AHP areas at the peak of
the agonist response. Ea: SP failed to reduce AHP area
in the presence of SR140333. Eb: SP (1 µM) was still
able to inhibit the AHP in the presence of SR142801. Ec:
senktide was still able to inhibit the AHP in the presence of SR140333.
However, the senktide-induced inhibition of the AHP was reduced by
SR142801.
