1Department of Pharmacology and Experimental Therapeutics and 2Department of Obstetrics, Gynecology, and Reproductive Science, University of Maryland School of Medicine, Baltimore, Maryland 21201
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ABSTRACT |
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Oh, Eun Joo,
Loren P. Thompson, and
Daniel Weinreich.
Sexually Dimorphic Regulation of NK-1 Receptor-Mediated
Electrophysiological Responses in Vagal Primary Afferent Neurons.
J. Neurophysiol. 84: 51-56, 2000.
Neurons can display sexual dimorphism in receptor expression,
neurotransmitter release, and synaptic plasticity. We have detected sexual dimorphism in functional tachykinin receptors in vagal afferents
(nodose ganglion neurons, NGNs) by studying the effects of hormonal
variation on the depolarizing actions of substance P (SP) in female
guinea pig NGNs. Using conventional "sharp" microelectrode recording plus measurement of serum 17-estradiol values, we examined SP responses in NGNs isolated from 1) ovariectomized females
(OVX), 2) OVX females treated with 17
-estradiol (OVX + E2), 3) pregnant females, and 4) males. Depending
on various manipulations, 19-41% female NGNs were depolarized
(16 ± 1.1 mV, mean ± SE) by 100 nM SP acting
through NK-1 receptors. The NGNs of OVX + E2 females (41%, 15/37;
17 ± 2.1 mV) and pregnant females (41%, 32/79; 16 ± 1.7 mV) were more likely to respond to SP than those of control males
(P < 0.001). The percentage of SP-sensitive NGNs from
OVX females (19%, 21/109; 15 ± 1.9 mV) was not significantly
different (P = 0.361) from that of control males (13%,
11/83; 13 ± 2.0 mV). The serum 17
-estradiol values for OVX + E2, pregnant, and OVX females were 23.9 ± 3.3 pg/ml
(n = 8), 16.0 ± 2.4 pg/ml (n = 4), and 3.9 ± 0.3 pg/ml (n = 8), respectively.
These data indicate that there is a gender difference in NK-1 receptor
expression in guinea pig nodose neurons, and they suggest that estrogen
may modulate SP responsiveness in these neurons.
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INTRODUCTION |
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Activation of estrogen receptors, which are
widely distributed throughout the central and peripheral nervous
systems (Bettini et al. 1992; Papka et al.
1997
), is critical for the development of sexual dimorphism.
Estrogen can also produce numerous neuromodulatory effects. In the CNS,
estrogen modulates the firing patterns and synaptic connectivity of
hypothalamic neurons involved in reproductive behavior in female
rats (Wong and Moss 1992
). In the peripheral nervous
system, the neuronal activity and receptive field size of
afferent fibers innervating the uterus show variation following the
estrous cycle (Robbins et al. 1992
). The effects of
estrogen on the nervous system are not only limited to areas obviously related to reproductive behavior. For example, following estrogen treatment of female rats, the size of facial mechanoreceptive fields of
trigeminal neurons is increased (Bereiter and Barker 1975
). Additionally, there are numerous reports of gender
differences in pain susceptibility and the response to analgesic
agents, such as morphine (Baamonde et al. 1989
;
Cicero et al. 1996
). These latter observations support a
role for estrogen as a modulator of sensory processing.
Estrogen also increases gene expression of numerous
peptides (Watters and Dorsa 1998) and receptors
(Gazzaley et al. 1996
), including neurokinin [substance
P (SP)] receptors (Villablanca and Hanley 1997
). SP is
released from peripheral nerve endings of small diameter afferents
eliciting peripheral reactions characterized as neurogenic inflammation
(Lembeck and Holzer 1979
). SP is also released centrally
from primary afferent nerve endings in the spinal dorsal horn, where it
contributes to the transmission of nociceptive information (De
Koninck and Henry 1991
). It has been recently demonstrated that
SP is released from the cell bodies of primary afferent neurons
(Huang and Neher 1996
). Primary afferent neurons not
only release SP, but some (dorsal root ganglion neurons) are also
excited by SP (Dray and Pinnock 1982
; Inoue et
al. 1995
; Li and Zhao 1998
; Spiegelman
and Puil 1990
) while others [nodose ganglion neurons (NGNs) of
the ferret] are inhibited by this neuropeptide (Jafri and
Weinreich 1996
, 1998
). These actions presumably
reflect the existence of SP autoreceptors in membranes of primary
afferent neurons.
We have reported that most adult primary vagal afferent somata from
male guinea pig are electrophysiologically unresponsive to exogenously
applied SP. However, following allergen-induced inflammation in vitro
(Weinreich et al. 1997) or in vivo (Moore et al.
1999b
), ~80% of NGNs exhibit an NK-2 receptor-mediated depolarizing response to bath-applied SP. In a preliminary series of
experiments with NGNs from female guinea pigs, we noted that, in
contrast to male NGNs, significant numbers of control NGNs were
depolarized by SP. Therefore, expression of tachykinin receptors in
guinea pig NGNs may be sexually dimorphic.
To determine whether estrogen regulates SP responsiveness of guinea pig
NGNs, we examined four groups of animals with various estrogen levels:
1) ovariectomized females (OVX), 2) OVX females with 17-estradiol treatment (OVX + E2), 3) near-term
pregnant females, and 4) control males. Our results reveal a
gender difference in the expression of functional NK-1 tachykinin
receptors in guinea pig NGNs.
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METHODS |
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Animals
Reproductively mature female Hartley guinea pigs (4-6 mo old,
bred at the University of Maryland) were anesthetized with ketamine (80 mg/kg ip)/xylazine (1 mg/kg im) and surgically ovariectomized through
bilateral flank incision. After at least 100 days, some ovariectomized
animals were anesthetized, and 21-day continuous-release pellets
containing 0.25 mg 17-estradiol (Innovative Research of America,
Sarasota, FL) were inserted in the neck subcutaneously. Twenty days
after the insertion of pellets, animals were anesthetized with
ketamine/xylazine, and both nodose ganglia were removed. Nodose ganglia
from ovariectomized animals not treated with estrogen were used as the
control group for 17
-estradiol-treated female animals. Near-term
pregnant guinea pigs (~60 days gestation; term, 65 days) were used as
the pregnant animal group. A subset of male guinea pigs (2-5 mo old)
was subject to castration to examine the contribution of testosterone
to the regulation of SP responsiveness. Guinea pigs were anesthetized
as described above, to allow the removal of both testicles through
bilateral scrotal incisions. These animals were allowed to recover at
least 7 days prior to removal of nodose ganglia. Ganglia from
noncastrated male guinea pigs served as controls. The University of
Maryland Institutional Animal Care and Use Committee approved all
methodology used in these experiments.
Tissue preparation and cell culture
Dissected ganglia were cleaned of adhering connective tissue and
debris in ice-cold 4°C Lockes solution (in mM): 136 NaCl, 5.6 KCl,
1.2 MgCl2, 2.2 CaCl2,14.3
NaHCO3, 1.2 NaH2PO4, and 10 dextrose,
equilibrated with 95% O2-5%
CO2, pH 7.2-7.4. Isolated NGNs were prepared
following the procedure outlined by Moore et al.
(1999a), except ganglia were kept at 4°C for 6-8 h with
collagenase (10 mg/ml) and dispase (10 mg/ml) in Hank's balanced salt
solution then incubated in the same solution at 37°C for 15 min. NGNs
were subsequently dissociated by trituration with fire-polished Pasteur pipettes of diminishing diameters. Isolated NGNs were resuspended in
Leibovitz L-15 medium (GIBCO BRL, Rockville, MD) containing 10%
(vol/vol) fetal bovine serum (FBS; JRH Biosciences, Lexena, KS). Cell
suspensions (0.15 ml) were transferred onto circular poly-D-lysine (Sigma Chemical, St. Louis, MO)-coated glass
coverslips (Bellco, Vineland, NJ) in a 24-well culture plate and
maintained at 37°C. NGNs were held in culture for 2-48 h prior to
electrophysiological recording. Coverslips were transferred to the
recording chamber and superfused with Lockes solution at 33-37°C.
Measurement of serum 17-estradiol
Arterial blood samples from anesthetized female animals were
collected in polypropylene tubes and centrifuged at 4,000 rpm at 4°C.
The serum supernatant was removed and stored at 20°C until assayed.
Serum 17
-estradiol was measured by the Coat-A-Count methodology
according to the manufacturer's instructions (Diagnostics Products,
Los Angeles, CA).
Electrophysiology and drug applications
Electrical properties and responses to SP were recorded
intracellularly using conventional "sharp" glass micropipettes
having DC resistances of 30-100 M when filled with 3 M KCl.
Current- and voltage-clamp recordings were made with an Axoclamp-2A
amplifier (Axon Instrument, Foster City, CA) in either bridge
(bandwidth 10 kHz) or discontinuous mode (sample rate 5 kHz, bandwidth
0.3-3 kHz). In discontinuous mode, the headstage voltage was
continually monitored to ensure that the sampled voltage reached steady
state. Membrane input resistance (Rin)
was monitored by measuring changes in the amplitude of electrotonic
voltage transients produced by 100-pA hyperpolarizing current pulses,
200 ms in duration. Neurons were included in this study if they had a
stable resting membrane potential (less than
50 mV), action potential
overshoot >20 mV, and a Rin > 20 M
. Data acquisition and analysis were performed with pClamp 6.2 software and a Digidata 1200 interface (Axon Instruments).
The recording chamber was mounted on the stage of a compound microscope equipped with Hoffman optics (×400) to visualize neurons for intracellular impalement. Coverslips in the recording chamber were superfused with Lockes solution via a gravity flow system (2-3 ml/min). The bath level was lowered to <50 µm above the neurons using an adjustable aspirator to minimize electrode stray capacitance. A concentration of 100 nM SP was used throughout this study because it evoked robust responses (>10 mV depolarization) but produced minimal desensitization with repeated exposure. SP was bath applied for 30 s, and antagonists were bath applied at least 3 min prior to the re-application of SP. When SP was administered multiple times, at least 3 min was allowed between applications.
Drug solutions
Drug solutions were prepared daily from concentrated stock solutions (1 mM) by dilution into Lockes solution. (+)-(2S,3S)-3-(2-methoxybenzylamino)-2-phenylpiperidine (CP99,994) was provided by Pfizer (Groton, CT), (S)-N-methyl-N[4-(4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl) butyl]benzamide (SR48,968) by Zeneca (Wilmington, DE), and SB 223412-A by SmithKline Beecham (Philadelphia, PA). All other drugs were purchased from Sigma Chemical.
Statistics
Data are expressed as means ± SE. Z-test was used to compare the percentage of neurons responding to SP, and one-way ANOVA was used to compare response characteristics among the groups. P < 0.05 was considered significant. Statistical analyses were performed using Sigmastat software (Jandel Scientific, San Rafael, CA).
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RESULTS |
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SP responsiveness and serum 17-estradiol level
Most NGNs from adult male guinea pig nodose ganglia do not show
electrophysiologically detectible responses to bath applied 100 nM SP
(Moore et al. 1999a,b
; Weinreich et al.
1997
). By contrast, 41% of acutely isolated NGNs from
ovariectomized and estrogen treated female guinea pig (OVX + E2, 6 animals) were depolarized an average of 17 mV by 100 nM SP
(Table 1 and Fig.
1). Some of the neurons fired action
potentials during the rising phase of the depolarization (Figs.
1C and 2). The traces in Fig.
1A illustrate a typical SP response recorded in the same
neuron in either current clamp or voltage clamp. Both responses were
accompanied by an increase in membrane conductance. During the peak of
the SP response, Rin fell to about
one-third of the value recorded before SP application (see Table 1).
The serum 17
-estradiol values in OVX + E2 females averaged 23.9 ± 3.3 pg/ml (mean ± SE, n = 8). SP responses
were also observed in NGNs prepared from pregnant females (9 animals; Fig. 1B). There were no significant differences in the
magnitude of the membrane depolarization, change in
Rin, duration of response (P > 0.1) as well as percentage of SP responsive
neurons (P = 0.842) between pregnant and the OVX + E2
females (Table 1). The serum 17
-estradiol values in pregnant females
averaged 16.0 ± 2.4 pg/ml (n = 4). For
comparison, we examined SP responses in NGNs from OVX females (9 animals) and from control males (11 animals). The serum level of
17
-estradiol was 3.9 ± 0.3 pg/ml (n = 8) in OVX females. The percentage of SP responsive neurons in these two
groups was significantly (P < 0.005) lower than in the
groups with elevated serum estrogen (OVX + E2 and pregnant females). Only 19 and 13% of OVX animals and control males, respectively, were
depolarized by SP (Table 1). However, the magnitude of the responses,
their duration, and changes in Rin
produced by SP were similar to values observed in NGNs from animals
with elevated serum estradiol (Table 1). There was no significant
difference in the percentage of SP responsive NGNs (P = 0.361) between OVX females and control males. Furthermore, several
passive and active membrane properties of NGNs were not significantly
different (P > 0.186-0.736) among four groups (Table
2).
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Pharmacological characterization of NK receptor type
The time course and magnitude of the SP responses, recorded in
either current or voltage clamp in NGNs from female guinea pigs (and
the responses in male guinea pigs, Fig. 1C), appeared similar to "unmasked" NK-2 receptor-mediated SP responses recorded in male NGNs following allergic inflammation (Weinreich et al. 1997) or exogenous application of serotonin (5-HT)
(Moore et al. 1999a
). To determine the nature of the
tachykinin receptor subtype underlying SP responses in female NGNs, we
treated NGNs from all groups with selective NK-1, NK-2, or NK-3
receptor antagonists (100 nM); CP99,994, SR,48,968, or SB223412-A,
respectively. SP responses from all animal groups, including control
male NGNs, were reversibly blocked by 100 nM CP99,994 applied prior to
and during SP application. The sample records in Fig. 2, A
and C, show the reversible block by CP99,994 recorded in a
NGN from OVX + E2 female and a control male NGN. SR48,968 and
SB223412-A did not block SP responses (Fig. 2B) from any
group. Taken together, these data suggest that NK-1 receptors mediate
SP responses in NGNs from control males and females, with high and low
serum estrogen concentrations.
Effect of testosterone on SP responsiveness
The percentage of SP responsive NGNs from control males and females with low serum estrogen levels were comparable (Table 1). To rule out a contribution of testosterone on SP response rate in males, we tested SP responsiveness in orchiectomized male animals (3 animals, 57 neurons). NGNs dissociated 1 wk after orchiectomy did not show any difference in response rate (18%, 10/57), receptor type (SP responses were blocked by CP99,994), response amplitude (17 ± 3.1 mV), response duration (69 ± 15.1 s), or membrane conductance change (38 ± 9.3%). This suggests that lower levels of testosterone in females, compared with male guinea pigs, do not contribute to their increased responsiveness to SP.
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DISCUSSION |
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Our primary observation is that elevated serum 17-estradiol,
achieved either by pregnancy or by chronic administration of estrogen,
directly or indirectly up-regulates NK-1 receptor-mediated SP responses
in female primary vagal afferent neurons. These results suggest that
estrogen can modulate sensory processing through regulation of
functional receptor expression.
Estrogen can have both acute and chronic effects on nervous tissues.
Acute effects occur within seconds to a few minutes after estrogen
exposure and disappear within seconds of estrogen removal. For example,
bath applied 17-estradiol reversibly potentiates kainate-induced
currents in acutely dissociated hippocampal CA1 neurons within 3 min
(Gu and Moss 1996
). This potentiation was mimicked by
8-bromo-cAMP and enhanced by a phosphodiesterase inhibitor, suggesting
that this action of 17
-estradiol is mediated by a second messenger,
in particular, cAMP. Membrane estrogen receptors or cytosolic
components have been suggested as possible mechanisms for these acute
effects (Moss et al. 1997
; Woolley 1999
).
Classically, estrogen affects neurons through genomic mechanisms.
Estrogen can diffuse across the plasma membrane of neurons and bind to specific intracellular receptors. These hormone-receptor complexes enhance the transcription of many genes and induce the synthesis of
specific proteins including membrane ligand-activated receptors. Genomic mechanisms usually requires hours to days. In the present study, we treated guinea pigs chronically with 17
-estradiol for 21 days, a time period sufficient for estrogen to exert genomic effects on
neurons and at a dose that produces serum levels similar to that
measured during pregnancy.
There are at least three possible mechanisms through which estrogen
might regulate the SP responsiveness of NGNs. First, some NGNs
innervating the uterus may respond to SP following estrogen-induced changes in the uterus. A subpopulation of NGNs innervates the uterus
(Ortega-Villalobos et al. 1990), and their peripheral
nerve terminals show diverse changes following the estrous cycle in the
rat (Robbins et al. 1992
). Signal molecules could thus
travel in vagal afferent axons from the uterus to the NGNs and trigger expression of NK responses. Although this may be a plausible
explanation for the expression of SP responses in some NGNs, the number
of SP responsive neurons in OVX + E2 and pregnant females (41%) are likely to exceed the number of NGNs innervating the uterus.
Second, estrogen may up-regulate SP responsiveness by altering the
animal's susceptibility to inflammation. It is well known that immune
responses can be influenced by sex hormones (Grossman 1984). In general, females have stronger humoral and
cell-mediated immunity. A more vigorous immune response not only makes
females more resistant to infections, but it also exaggerates responses to autoantigens and can induce autoimmune disease (Ansar Ahmed et al. 1985
). In addition, estrogen contributes to
hypersensitivity reactions such as allergic rhinitis and asthma by
augmenting mast cell degranulation (Vliagothis et al.
1992
), and by up-regulation of histamine receptors
(Hamano et al. 1998
). Thus tachykinin receptors might also be up-regulated secondary to activation of immune cells. Although tractable, this explanation is in conflict with results from
our previous studies. Following immunological activation of mast cells
in vitro (Weinreich et al. 1997
) or in vivo
(Moore et al. 1999b
), functional NK-2 receptors, not
NK-1 receptors, are expressed in male guinea pig NGNs.
Last, and most plausibly, estrogen may directly act on estrogen
receptors in NGNs, leading to increased SP responsiveness. Estrogen
might simply increase the number of SP receptors by enhancing transcriptional processes or trigger the translocation of SP receptors from cytosol to the cell surface. It is also possible that estrogen activates or augments a regulator component of the SP receptors without
changing the number of receptors. This interpretation is supported by
the finding that estrogen receptor protein and mRNA are localized in
the rat NGNs (Papka et al. 1997) and that physiological
serum levels of 17
-estradiol correlate with the percentage of
neurons expressing functional NK-1 receptor-mediated responses.
Finally, preliminary data reveals that 17
-estradiol can increase SP
responsiveness in isolated NGNs. Eleven of 36 NGNs incubated with 50 nM
17
-estradiol for >30 min showed SP response (14 ± 4.2 mV),
while only 3/30 NGNs treated with vehicle responded.
In summary, we have shown that gender differences in the SP responses in guinea pig vagal afferent neurons are due to a sexually dimorphic expression of functional NK-1 receptors. Differences in estrogen levels are most likely responsible for the higher percentage of SP-responsive NGNs in females. These results illustrate that estrogen can modulate sensory processing via control of functional tachykinin receptor expression in primary afferent neurons.
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ACKNOWLEDGMENTS |
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The authors thank E. Lancaster and Drs. K. A. Moore and M. S. Gold for helpful comments on an earlier version of this manuscript. We also thank G. Pinkas for technical assistance.
This work was supported by National Institute of Health Grants NS-22069 to D. Weinreich and HL-49999 to L. P. Thompson.
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FOOTNOTES |
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Address for reprint requests: D. Weinreich, Dept. of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Rm. 4-002, Bressler Research Building, 655 West Baltimore St., Baltimore, MD 21201-1559 (E-mail: dweinrei{at}umaryland.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 13 December 1999; accepted in final form 16 March 2000.
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REFERENCES |
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