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Department of Pharmacology, University of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania 15261
Submitted 5 February 2003; accepted in final form 21 March 2003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Ala8]-neurokinin A (4–10), enhanced
Ca2+ currents (5–36% increase, 0.05–0.5
µM). The enhancement was reversed by the NK2 antagonist MEN
10,376 (0.2 µM) but unaffected by the NK3 antagonist SB 235,375
(0.2 µM). The NK3 agonist [MePhe7]-neurokinin B
(0.5–1.0 µM) inhibited Ca2+ currents
(6–24% decrease). This inhibition was not prevented by the
NK2 antagonist MEN 10,376 (0.2 µM) but was blocked by the
NK3 antagonist SB 235,375 (0.2 µM). Both the enhancement and
inhibition of Ca2+ currents by neurokinin agonists were
reversed by the protein kinase C inhibitor bisindolylmaleimide I HCl
(0.2–0.5 µM). Following inhibition of Ca2+
channels by [MePhe7]-neurokinin the facilitatory effect of BayK
8644 (5 µM) was increased and the inhibitory effect of the N-type
Ca2+ channel blocker w -conotoxin GVIA (1 µM) was
diminished, suggesting that the NK3 agonist inhibits N-type
Ca2+ channels. Similarly, block of all but N-type
Ca2+ channels, revealed that
[Ala8]-neurokinin A (4–10) enhanced the currents while
[MePhe7]-neurokinin B inhibited the currents. Inhibition of all but
L-type Ca2+ channels, revealed that
[Ala8]-neurokinin A (4–10) enhanced the currents while
[MePhe7]-neurokinin B had no effect. Activation of protein kinase C
with low concentrations of phorbol-12,13-dibutyrate enhanced
Ca2+ currents, but high concentrations inhibited N- and
L-type Ca2+ currents. In summary, these data suggest
that in adult rat dorsal root ganglia neurons, NK2 receptors
enhance both L- and N-type Ca2+ channels and
NK3 receptors inhibit N-type Ca2+ channels
and that these effects are mediated by protein kinase C phosphorylation of
Ca2+ channels. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Marco et al.
1998; Otsuka and Yoshioka
1993; Zogorodnyuc and Maggi
1997), contribute to numerous physiological processes, including
inflammatory responses (Jia and Seybold 1996), nociception
(Liu and Sandkuhler 1997),
central and peripheral cardiovascular regulation
(Thompson et al. 1998),
urinary bladder function (Kiss et al.
2001; Tramontana et al.
1998), and other autonomic reflexes
(Moodley et al. 1999;
Serio et al. 1998). The role
of neurokinins in central and peripheral nociceptive mechanisms has been
examined in considerable detail (Cheunsuang
and Morris 2000; Hunt
2000; Laird et al.
2000; McLeod et al.
1999). Thermal hyperalgesia associated with neuropathic and
inflammatory pain is reduced after ablation of substance P-containing spinal
neurons with substance P-saporin (Nichols
et al. 1999). Knockout mice lacking functional NK1
receptors show deficits in perception of visceral pain (intracolonic
capsaicin) and do not develop hyperalgesia and edema in response to noxious
stimuli (Laird et al. 2000).
On the other hand, transgenic mice overexpressing substance P show
hyperalgesia that is reversed by substance P antagonists
(McLeod et al. 1999). It is
also important to note that mice with a deletion of the preproneurokinin A
gene, which codes for both substance P and neurokinin A, lack several
responses associated with noxious stimuli (e.g., plasma extravasation) and
fail to detect the normal range of intensities of nociceptive stimuli,
although they respond normally to acute pain tests
(Basbaum 1999). These mice also
exhibit impaired responses to chemical irritation of the urinary bladder
(Kiss et al. 2001).
Different neurokinins preferentially activate three types of receptors:
substance P for NK1, neurokinin A for NK2, and
neurokinin B for NK3 (Maggi
1995). There is evidence for expression of all three types of
receptors in dorsal root ganglia neurons, although they may be differentially
expressed in the cell bodies and terminals of these neurons
(Brechenmacher et al. 1998;
Hu et al. 1997;
Malcangio and Bowery 1999;
Schmid et al. 1998). More
selective neurokinin receptors agonists and antagonists have been developed
recently and allow for more precise characterization of the receptors involved
in various physiological responses (Lippe
et al. 1997; Maggi and
Schwartz 1997; Matuszek et al.
1998; Torrens et al.
1997).
In addition to mediating transmission between primary afferent neurons and
peripheral target organs or second order neurons in the CNS, neurokinins may
also act on autoreceptors on afferent nerve terminals to regulate terminal
excitability (Wen and Morrison
1996) or transmitter release
(D'Agostino et al. 2000;
Malcangio and Bowery 1999).
Neurokinin autoreceptors are thought to be linked through G proteins to
protein kinase C (and/or other protein kinases), which in turn can
phosphorylate and modulate Ca2+ channels
(Hall et al. 1995;
Harding et al. 1999;
Murase et al. 1989;
Schmid et al. 1998). L-type
Ca2+ channels have been implicated in neuropeptide
release from sensory neurons (Harding et
al. 1999; Holz et al.
1988; Perney et al.
1986; Rane et al.
1987; Yoshizawa et al. 1989), whereas N-type
Ca2+ channels may be involved in the release of glutamic
acid (Gruner and Silva 1995; Winkler
1997). SP or protein kinase C are known to enhance
Ca2+ currents in many cell types
(Mayer et al. 1990;
Murase et al. 1989;
Sims et al. 1988;
Womack et al. 1986), including
dorsal root ganglia neurons (Hall et al.
1995). However, activation of protein kinase C has also been
reported to inhibit Ca2+ currents
(McMahon et al. 2000). Thus
the effect and identity of neurokinin auto-receptors subtypes and the
signaling mechanisms activated by neurokinin receptors to modulate
Ca2+ channels in dorsal root ganglia neurons requires
further study.
In the present experiments, we examined the effect of three types of neurokinin receptor agonists and antagonists on Ca2+ currents in dissociated dorsal root ganglia neurons from adult rats. Our data suggest that activation of NK2 receptors enhances both L-type and N-type Ca2+ currents, while activation of NK3 receptors inhibits N-type Ca2+ currents. These responses are antagonized by the selective protein kinase C-inhibitor, bisindolylmaleimide, suggesting that they are mediated by activation of protein kinase C.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Cell preparation
Single cells were isolated by standard enzymatic techniques as previously
described (Sculptoreanu et al.
1995). Freshly dissected ganglia were minced and washed in cold,
oxygenated DMEM (Sigma). This was followed by a brief, 10-min dissociation at
37°C in DMEM containing 0.5 mg/ml trypsin (Sigma). After a 10-min
centrifugation, the media was replaced with DMEM containing 1 mg/ml
collagenase B (Boehringer-Mannheim) and 0.5 mg/ml trypsin inhibitor type 1S
(Sigma). Dissociation of neurons was monitored and every 10 min the cells
gently triturated with siliconized Pasteur pipettes. After the ganglia were
dissociated into individual neurons (25–40 min), the cell suspension was
centrifuged for 10 min at 1,200 rpm. The pellet was layered on 20 ml of 50%
adult bovine serum (Sigma) and DMEM and centrifuged again at 800 rpm. This
step removed most of the debris and broken cells. The pellet was resuspended
in DMEM containing 10% heat inactivated horse serum and 5% fetal bovine serum
(Sigma) and plated on collagen coated 35 mm petri dishes (Collaborative
Research, Biocoat). Neurons were plated at low density (2,000–3,000 per
dish). Primary cultures were kept in a 95% O2–5%
CO2 incubator at 37°C.
Recording
The whole cell patch-clamp technique was used to record Ca2+ currents in dorsal root ganglia neurons. Patch pipettes were pulled from capillary glass tubes (Accufil 90, Clay-Adams) on a horizontal puller (Model P8 PC, Sutter Instruments) and fire polished. Immediately before recording, the serum containing media was replaced with Ca2+ containing solutions. Whole cell currents were recorded using an Axopatch 1D (Axon Instruments) patch-clamp amplifier. Pulse generation, current recording, and data analysis used pClamp software (Axon Instruments). Curve fitting was done using Sigma Plot software (Jandel Scientific). Currents were sampled at 50–500 ms, filtered at 2 kHz, and capacitative current and ≤80% of the series resistance was compensated. A p/4 protocol was used to subtract uncompensated capacitative currents and leak currents.
For whole cell recordings, modified physiological solutions were used to
suppress Na+, K+, and Cl– channel currents. The
pipette (intracellular) solution consisted of (in µM) 130
N-methyl-D-glucamine, 20 EGTA (free acid), 5 BAPTA, 10
HEPES, 6 Mg(OH)2, 4 Ca(OH)2, 3 (Mg)ATP, 0.3 (Li) GTP,
and 0.3 (Na) cAMP, pH buffered to 7.3 with methanesulfonic acid. The external
solution contained (in µM) 20 Ca(OH)2, 60 tetraethyl ammonium
(OH), 40 tris [hydroxyethyl] aminomethane (Trizma base), 5 4-aminopyridine,
and 10 HEPES, pH buffered to 7.4 with methane-sulfonic acid. In these
recording solutions, Ca2+ currents run-up during the
initial 1–3 min of recording
(Sculptoreanu et al., 1995).
Drugs were added after recording stable currents for an additional 1–3
min.
Pharmacology
Various drugs were used to separate Ca2+ channel
currents: 
-conotoxin GVIA as an N-type blocker, Bay K 8644 and
nitrendipine as agonist and blocker of L-type Ca2+
currents (Sigma), -agatoxin IVA and agatoxin TK (Sigma) for P type,
-conotoxin MVIIC for Q-type, and flurenzipine (Calbiochem) for T-type
Ca2+ currents. The following neurokinin agonists and
antagonists were used: substance P, the NK1 agonist
[Sar9, Met11]-substance P, the NK2 agonist
[Ala8]-neurokinin A (4–10), the NK3 agonist
[MePhe7]-neurokinin B (Calbiochem), the NK1 antagonist
[Tyr6, Phe7, D-His9]-Substance P
(sentide, Peninsula Labs), the general neurokinin receptor antagonist
[D-Pro2, D-Phe7,
D-Trp9]-substance P, and the NK2 antagonist
MEN 10,376 (Sigma). Neurokinin agonists and antagonists were prepared in
aqueous solutions. The NK3 receptor antagonist SB 235,375 was a
generous gift from SmithKline Beecham. The phorbol ester, phorbol
12,13-dibutyrate (Research Biochemicals), and the PKC inhibitor
bisindolylmaleimide I HCl (Calbiochem) were dissolved in DMSO (100 µM) and
used at <0.01% of their stock concentration. At these dilutions, DMSO alone
had no effect on calcium channel currents. Stock solutions in 10–100
µM were stored at –20°C and diluted in the external recording
solution just before experiments. Extracellularly applied drugs were pipetted
from stock solutions at 10 to 100 times the final concentration and rapidly
mixed in the recording chamber as described previously
(Sculptoreanu et al. 1995).
Steady-state effects of each drug concentration were measured for
≥2–3 min before changing the drug concentration or adding a new
drug.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Yoshimura et al. 2001), small to medium size rat dorsal root
ganglia cells exhibit both N-type and L-type Ca2+
currents. -Conotoxin GVIA (1 µM), an N-type
Ca2+ channel blocker, reduced the amplitude of
Ca2+ currents by 51 ± 5% (n = 21,
Table 1), whereas nitrendipine
(5 µM), an L-type channel blocker, reduced the currents by 34 ± 6%
(n = 17); 23 ± 9% of the current remained after combined
-conotoxin GVIA-nitrendipine treatment (n = 14). These
remaining currents were reduced to 12 ± 8% of control currents by a
combination of Q- and P-type channel blockers (-conotoxin MVIIC, 0.5
µM, n = 5 and -agatoxin IVA, 0.1 µM, n = 5 or
-agatoxin TK, 0.1 µM, n = 4). Bay K 8644 (5 µM), a
dihydropyridine agonist that is known to enhance L-type
Ca2+ currents
(Sculptoreanu et al. 1995),
consistently enhanced the currents in either untreated neurons or after the
application of other agents such as -conotoxin GVIA or neurokinins
(Table 1).
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Dual effect of substance P in dorsal root ganglia neurons
The effects of substance P and other agonists and inhibitors were studied
with cumulative addition of drugs (Fig.
1). A test pulse near the peak of the current voltage relationship
of Ca2+ currents (0 mV), 50 or 500 ms in duration, was
repeated at 5- to 20-s intervals. Drugs were added to the bath and rapidly
mixed. At most, three concentrations of substance P were tested on each cell.
Changes in currents occurred within 2 min or less after each concentration was
tested, and steady-state effects were measured for 1–2 min prior to
raising drug concentrations (Fig.
1). Preliminary experiments revealed that substance P in a low
concentration (<0.2 µM) facilitated Ca2+ currents
(9–35% increase, n = 10,
Fig. 1A). In
subsequent experiments (n = 35 neurons), several concentrations of
substance P were added to the bath at 5- to 10-min intervals to construct
cumulative concentration response curves (Figs.
1 and
2). Various concentrations of
substance P were tested (in µM: 0.05, n = 25; 0.1, n =
14; 0.2, n = 23; 0.5, n = 12; 1.0, n = 4).
Substance P concentrations between 0.05 and 0.2 µM enhanced
Ca2+ currents (5–40% increase), whereas at
concentrations of substance P above 0.5 µM, the enhancements were
progressively reduced (Fig.
2A, 
). This reduction in Ca2+
current enhancements at higher substance P concentrations was abolished in the
presence of SB 235,375 (0.2 µM), an NK3 antagonist
(Hay et al. 2002), added
either before or after substance P (Fig.
1A or
2A, 
). Treatment
with SB 235,375 did not alter Ca2+ currents in the
absence of substance P (Fig.
2A) but did increase the currents (10–35%) in the
presence of substance P at all concentrations tested (0.05–1.0 µM,
n = 15 cells tested; Fig.
2A, ). For example, in the presence of 0.2 µM of
substance P, addition of SB 235,375 (0.2 µM) significantly increased
Ca2+ currents (26 ± 5%, n = 6,
P < 0.01, Figs.
1A and
2A). In the presence
of substance P and SB 235,375, application of -conotoxin GVIA markedly
reduced the currents; whereas Bay K 8644 facilitated the currents
(Table 1).
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In a separate series of experiments, the facilitatory effect of 0.2 µM substance P on Ca2+ currents was reversed by addition of the protein kinase C inhibitor bisindolylmaleimide (Figs. 1B and 2C, 0.2 µM, n = 4 and 0.5 µM, n = 14). The enhancement of Ca2+ currents by 0.2 µM substance P was also reversed by bath application of the general neurokinin receptor antagonist [D-Pro2, D-Phe7, D-Trp9]-substance P (Fig. 2C, SP Ant, 0.2 µM). Addition of bisindolylmaleimide (0.2 µM, n = 5) after [D-Pro2, D-Phe7, D-Trp9]-substance P had no additional effect on Ca2+ currents. These data suggest that the effect of bisindolylmaleimide was due a suppression of the action of substance P and not due to a direct inhibition of Ca2+ currents by bisindolylmaleimide.
Lack of effect of an NK1 agonist in dorsal root ganglia neurons
The NK1 agonist [Sar9, Met11]-substance P
(sar,metSP) did not alter Ca2+ currents in dorsal root
ganglia neurons (n = 13) at concentrations ranging between 0.05 and 1
µM. At most, three increasing concentrations were added in sequence for
each cell tested and currents were measured for 5–10 min for each
concentration. Current densities for control and after sar,metSP were
unchanged as follows: control (n = 9): 26.8 ± 2.7 pA/pF; 0.05
µM (n = 7): 27.4 ± 0.3 pA/pF; 0.1 µM (n = 9):
28.1 ± 0.5 pA/pF; 0.2 µM (n = 4): 26.9 ± 1 pA/pF;
0.5 µM (n = 8): 26.7 ± 0.3 pA/pF; 1.0 µM (n =
8): 26.3 ± 0.5 pA/pF. In another series of experiments, the PKC
inhibitor bisindolylmaleimide (0.5 µM, n = 3; 1.0 µM,
n = 4) had no effect after 0.2 µM [Sar9,
Met11]-substance P (0.5 µM). In these experiments current
densities were 28.3 ± 3.2 pA/pF in the control recordings and 25.9
± 2.7 pA/pF after the NK1 agonist [Sar9,
Met11]-substance P, and were not changed by bisindolylmaleimide
(0.5 µM, 25.9 ± 2.7 pA/pF; 1.0 µM, 26.3 ± 0.4 pA/pF). This
suggests that protein kinase C inhibitor was devoid of nonspecific effects on
Ca2+ currents and that dorsal root ganglia neurons have
little endogenous protein kinase C activity under the recording conditions
used here (Fomina and Levitan
1997).
NK2 receptors mediate an enhancement of Ca2+ currents in dorsal root ganglia neurons
Since the enhancement of Ca2+ currents by substance P
is not prevented by an NK3 antagonist or reproduced by an
NK1 agonist, it may be mediated by NK2 receptors. To
test this idea, we used [Ala8]-neurokinin A (4–10), a
selective NK2 agonist. [Ala8]-Neurokinin A
(4–10) increased Ca2+ currents at all
concentrations tested (0.05–0.5 µM,
Fig. 3, n = 12 cells
tested). The enhancement of Ca2+ currents (5–60%
increase) occurred within 2 min, and steady-state effects were measured for
1–2 min prior to raising drug concentrations. Administration of an
NK3 inhibitor, SB 235,375 (0.2 µM, n = 6; 0.5 µM,
n = 4), before or after 0.2 µM of the NK2 agonist did
not prevent the enhancement of currents by [Ala8]-neurokinin
A (4–10) (Fig. 3, A2 and
B). At most, three increasing concentrations of the drug
were added in sequence for each cell tested. In a separate series of
experiments, the NK2 receptor antagonist MEN 10,376 (0.2 µM)
reversed the enhancement of Ca2+ currents induced by 0.2
µM (n = 4) or 0.5 µM (n = 4)
[Ala8]-neurokinin A (4–10)
(Fig. 3, A3 and
B). In three of these experiments, after reversal of
enhancement by 0.2 µM [Ala8]-neurokinin A (4–10),
addition of 0.5 µM agonist in the presence of the NK2 antagonist
no longer had an effect. These data suggest that
[Ala8]-neurokinin A (4–10) is a specific agonist for
NK2 receptors and had little or no effect on NK3
receptors. Figure 3C
shows an additional experiment in which bisindolylmaleimide reversed the
stimulation of Ca2+ currents by 0.2 µM
[Ala8]-neurokinin A (4–10), which produced a 35%
enhancement. After stable currents were recorded
(Fig. 1), current-voltage
relationships were generated by a series of depolarizing pulses from –60
to +70 mV (Fig. 3C).
The enhancement of currents by the neurokinin agonist was not voltage
dependent. In eight additional cells, 0.5 µM bisindolylmaleimide (26
± 2 pA/pF, at 0 mV) reversed the enhancement of current densities after
the agonist ([Ala8]-neurokinin A (4–10), 0.2 µM, 35
± 6 vs. 26 ± 1 pA/pF for control currents). These data suggest
that the stimulatory effects of [Ala8]-neurokinin A
(4–10) are mediated by protein kinase C.
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NK3 receptors mediate an inhibition of Ca2+ currents in dorsal root ganglia neurons
The selective NK3 agonist [MePhe7]-neurokinin B inhibited Ca2+ currents (0.05–1.0 µM, Fig. 4). This effect of [MePhe7]-neurokinin B was reversed by SB 235,375 (0.2 µM, Figs. 4A3 and 2B, n = 6). At most, three increasing concentrations of [MePhe7]-neurokinin B were added in sequence for each cell tested, and currents were measured for at least 1 min after steady-state effect at each concentration tested. The inhibitory effect of 0.2 µM [MePhe7]-neurokinin B was enhanced from 10 to 26% by administering 0.2 µM of the NK2 antagonist, MEN 10,376 before [MePhe7]-neurokinin B (Fig. 4B, P < 0.001). These data suggest that this agonist may have weak NK2 effects. In a separate series of experiments, the protein kinase C inhibitor bisindolylmaleimide (0.5 µM, n = 6) reversed the inhibition of Ca2+ currents by 0.2 µM [MePhe7]-neurokinin B (Fig. 4C). In another series of experiments, application of bisindolylmaleimide (0.5 µM) before 0.2 µM of [MePhe7]-neurokinin B (n = 3) prevented the inhibitory effects (12% inhibition) of the NK3 agonist. After stable currents were recorded (Fig. 1), current-voltage relationships were generated by a series of depolarizing pulses from –60 to +70 mV (Fig. 4C). The inhibition of currents by [MePhe7]-neurokinin B was not voltage dependent. In six additional cells, control current densities were 30 ± 1 pA/pF. Additional NK3 0.2 µM agonist [MePhe7]-neurokinin B inhibited the currents (26.5 ± 0.9 pA/pF); 0.5 µM of bisindolylmaleimide reversed this inhibition to control magnitudes (29.8 ± 0.7 pA/pF). These data suggest the inhibitory action of the NK3 agonist is due to protein kinase C phosphorylation of Ca2+ channels.
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Phorbol 12,13-dibutyrate modulates L- and N-type Ca2+ currents in dorsal root ganglia neurons
The reversal of NK2- and NK3-mediated effects by the
protein kinase C inhibitor suggests that protein kinase C may participate in
both facilitatory and inhibitory modulation of Ca2+
currents. Figures 5 and
6 summarize experiments in
which we tested the effect of a protein kinase C activator, phorbol
12,13-dibutyrate (0.05–90 µM). Phorbol 12,13-dibutyrate both enhanced
and inhibited calcium currents (Fig.
5). Enhancement occurred at low concentrations (0.05–1.0
µM, n = 4, Figs.
5A and
6), and inhibition occurred at
higher concentrations (25–90 µM, n = 6, Figs.
5B and
6A). At most, five
increasing concentrations of phorbol 12,13-dibutyrate were added in sequence
for each cell tested (n = 22), and currents were measured for at
least 1 min after steady-state effect at each concentration tested. The
enhancement by 0.2 µM phorbol 12,13-dibutyrate was reversed by
bisindolylmaleimide (Fig.
6B; 0.2 µM, n = 4). In these cells
-conotoxin GVIA (1 µM, n = 6) or a combination of
-conotoxin GVIA and Bay K 8644 (n = 3) produced effects
similar to those in the previous series of experiments; notably, a >50%
inhibition of currents in response to the N-type Ca2+
channel blocker and a >40% enhancement by the L-type
Ca2+ channel agonist after -conotoxin GVIA block.
This suggests that these small dorsal root ganglia neurons expressed
relatively large levels of N- and L-type Ca2+
currents.
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To evaluate more directly the types of Ca2+ channels
affected by phorbol 12,13-dibutyrate, the effects of various antagonists were
tested. As shown in Fig.
6C, when Ca2+ currents were blocked
by a combination of P-type Ca2+ channel blocker
-agatoxin IVA (0.05 µM), L-type Ca2+ channel
blocker nitrendipine (5 µM), and T-type Ca2+ channel
blocker flurenzipine (200 µM), phorbol 12,13-dibutyrate (0.05 µM,
n = 5; 0.10 µM, n = 4; 0.20 µM, n = 6) still
enhanced the currents, and bisindolylmaleimide (0.2 µM, n = 6; 0.5
µM, n = 4) reversed the enhancement. The N-type
Ca2+ channel blocker -conotoxin GVIA blocked
between 49 ± 6% (1 µM, n = 4) and 89 ± 3% (10 µM,
n = 6) of these agatoxin-dihydropyridine-flurenzipine-insensitive
currents. Ca2+ currents blocked by a combination of
-agatoxin IVA (0.05 µM) and -conotoxin GVIA (1 µM) and
enhanced by 5 µM Bay K 8644 were also increased by 0.2 µM phorbol
12,13-dibutyrate (Fig.
6D, n = 6). This enhancement of L-type
Ca2+ currents was also reversed by 0.2 µM
bisindolylmaleimide. Therefore low concentrations of phorbol ester seem to
enhance both L- and N-type Ca2+ currents. In five cells
tested, after enhancement of Ca2+ currents by 0.2 µM
phorbol 12,13-dibutyrate, application of 0.2 µM
[Ala8]-neurokinin A (4–10) no longer had an effect
(data not shown). This suggests that the NK2 agonist, and low
concentrations of the phorbol 12,13-dibutyrate, may activate phosphorylation
of the same sites on Ca2+ channels.
Subtypes of Ca2+ channels are modulated selectively by neurokinins receptors
The interactions between the neurokinins and -conotoxin GVIA and Bay
K 8644 were also used to evaluate the types of Ca2+
channels affected by the neurokinins. We reasoned that if a neurokinin
selectively enhanced or suppressed N- or L-type Ca2+
channels, the magnitude of the effect of -conotoxin GVIA or Bay K 8644
would be changed in the presence of that neurokinin. As shown in
Table 1, the suppression of
Ca2+ currents by -conotoxin GVIA (approximately
50% suppression) was not changed in the presence of the inactive
NK1 analog, [Sar9, Met11]-substance P or in
the presence of substance P, substance P plus SB 235,375, or [
Ala8]-neurokinin A (4–10), which enhance
Ca2+ currents. We assume that if the facilitatory agents
selectively enhanced one Ca2+ channel, the percent
inhibition by -conotoxin GVIA would have been altered. Therefore it
seems likely that the facilitatory agents enhanced both types of
Ca2+ channels. On the other hand, in the presence of the
inhibitory effect of 0.2 µM [MePhe7]-neurokinin B, the
depressant effect of -conotoxin GVIA was significantly reduced (36 vs.
49% depression), indicating that the NK3 agonist has a greater
inhibitory effect on N-type versus L-type Ca2+
channels.
The facilitatory effect of BayK 8644 (approximately 25% increase in the
presence of the NK1 agonist) was not changed by substance P or by
the [Ala8]-neurokinin A (4–10) but was enhanced (34%
increase) in the presence of [MePhe7]-neurokinin B. This change is
also consistent with the view that the NK3 agonist suppresses
N-type Ca2+ currents, thereby increasing the relative
contribution of L-type Ca2+ currents to the total
current and thus increasing the magnitude of the BayK 8644–induced
facilitation. This idea was further tested in another series of experiments
(Fig. 7).
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As shown in Fig.
7A, the NK2 agonist
[Ala8]-neurokinin A (4–10) (0.5 µM) enhanced the
Ca2+ currents after block by 42% of all but N-type with
a combination of L-, Q-, and P-type Ca2+ blockers
(nitrendipine, 5 µM; -conotoxin MVIIC, 0.5 µM; -agatoxin
TK, 0.1 µM) and addition of NK3 receptor antagonist, SB 235,375
(0.5 µM, n = 5). Only about 8% of residual currents remained after
block of N-type Ca2+ with -conotoxin GVIA (1
µM). Conversely, the NK3 agonist [MePhe7]-neurokinin
B (0.5 µM, Fig. 7B,
n = 5) inhibited 37% of N-type Ca2+ currents
after block of L-, Q-, and P-type Ca2+ currents and
addition of NK2 antagonist, MEN 10,376 (0.5 µM). Only about 9%
of residual currents remained after block of N-type Ca2+
with -conotoxin GVIA (1 µM). [Ala8]-neurokinin A
(4–10) (0.5 µM) elicited a 52% increase in L-type
Ca2+ currents after block of N-, Q-, and P-type
Ca2+ currents with appropriate antagonists and addition
of the NK3 antagonist, SB 235,375 (0.5 µM,
Fig. 7B, n =
8). The L-type Ca2+ channel blocker nitrendipine (5
µM) blocked 87% of the [Ala8]-neurokinin
A–stimulated current. The NK3 agonist,
[MePhe7]-neurokinin B (0.5 µM), had a small inhibitory effect on
L-type Ca2+ currents after block of N-, Q-, and P-type
Ca2+ currents and addition of the NK2
antagonist, MEN 10,376 (0.5 µM, Fig.
7D, n = 6). However, this effect was not
statistically significant (P = 0.66). The L-type
Ca2+ channel blocker nitrendipine (5 µM) blocked 90%
of this current, indicating that it was mostly L-type
Ca2+ current (Fig.
7D). Insets in
Fig. 7 show control currents
after block of all but N-type Ca2+ currents with a
combination of blockers (Fig. 7, A
and B) and after [Ala8]-neurokinin A
(0.2 µM, Fig. 7A)
or [MePhe7]-neurokinin B (0.2 µM,
Fig. 7B) and
-conotoxin GVIA (1 µM). Insets in
Fig. 7, C and
D, show control currents after block of all but L-type
Ca2+ currents with a combination of blockers and after
[Ala8]-neurokinin A (0.2 µM,
Fig. 7C) or
[MePhe7]-neurokinin B (0.2 µM,
Fig. 7D) and
nitrendipine (5 µM). Note that the N-type currents in
Fig. 7, A and
B, typically activate and inactivate more rapidly than
the L-type currents in 7, C and D. The slow inactivation of
L-type Ca currents is apparent even when longer test pulses, 500 ms in
duration, are used (Fig.
7D).
|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Subtypes of neurokinin receptors that modulate Ca2+ channel activities in dorsal root ganglia neurons
The conclusion that NK2 and NK3 exert opposite
modulatory effects in dorsal root ganglia neurons is based on the complex
effects of substance P as well as on the effects of selective neurokinin
agonists and antagonists. For example, substance P enhanced
Ca2+ currents at concentrations below 0.2 µM, but at
higher concentrations the enhancement of the current gradually diminished
(Fig. 2). This might be
attributable to receptor desensitization during repeated administration of
substance P. However, this explanation seems unlikely because an
NK3 antagonist prevented the decline in the facilitatory response
and enhanced the facilitatory responses to high concentrations of substance P.
Thus facilitation of Ca2+ currents by low concentrations
of substance P and apparent reversal of facilitation by higher concentration
must be mediated by different receptors. The finding that low concentrations
of substance P produce facilitation and high concentrations reverse this
facilitation is consistent with the greater affinity of substance P for
NK2 versus NK3 receptors
(Torrens et al. 1997). The
failure to demonstrate receptor-mediated inhibition with sequential
application of increasing concentrations of substance P raises the possibility
that prior activation of NK2 receptors with low concentrations of
substance P might down-regulate the NK3-mediated inhibition or that
the NK3 receptors partially desensitize during prolonged exposure
to substance P.
The conclusions regarding the actions of substance P are supported by the
effects of more selective neurokinin agonists. The NK2-specific
agonist [Ala8]-neurokinin A (4–10) enhanced the
Ca2+ currents to a magnitude similar to that seen with
substance P after inhibition of NK3 receptors with SB 235,375. The
NK2 receptor antagonist MEN 10,376 reversed the enhancement of
Ca2+ currents by [Ala8]-neurokinin A
(4–10). On the other hand, the selective NK3 agonist
[MePhe7]-neurokinin B inhibited Ca2+
currents, and this effect was blocked the NK3 receptor antagonist.
Thus various evidence indicates that two types of neurokinin receptors with
opposing effects on Ca2+ channels can be expressed in
the same dorsal root ganglia neuron. The experiments in
Fig. 7 demonstrate that, while
NK2 receptor activation facilitates both N- and L-type
Ca2+ currents, activation of NK3 receptors
mainly inhibits N-type Ca2+ currents.
Although other studies have identified inhibitory effects of neurokinins on
ion channels in various types of neurons, this is the first report of a
neuronal inhibitory action that can be attributed to NK3 receptors.
Substance P was shown to inhibit Ca2+-dependent
K+-channels and other K+ channels
(Gilbert et al. 1998;
Otsuka and Yoshioka 1993;
Phenna et al. 1996); however,
the subtype of neurokinin receptor involved was not established. In nucleus
basalis cholinergic neurons, substance P inhibits N- but not L-type
Ca2+ channel currents
(Margeta-Mitrovic et al.
1997), but again the type of receptor mediating this effect is not
known. On the other hand, NK3 receptors have been implicated in
neuronal excitatory effects mediated by activation of nonspecific cation
channels (Hardwick et al.
1997; Hu et al.
1997). Substance P released from the soma or terminals of dorsal
root ganglia neurons may thus facilitate its own release by activation of
nonselective cation channels that are partially permeable to calcium ions
(Hu et al. 1997;
Schmid et al. 1998). Indeed,
it has been reported that activation of NK3 receptors can enhance
the K+-evoked release of substance P from capsaicin-sensitive
synaptosomes obtained from the rat spinal cord
(Schmid et al. 1998). These
data suggest that NK3 receptors might have a facilitatory
autoreceptor function at central afferent terminals, whereas our data would
suggest the opposite effect. Clearly, the significance of this difference
between the two series of experiments will have to be explored in further
studies.
Protein kinase C is the mediator of NK2 and NK3 receptor action on Ca2+ currents
The enhancement of Ca2+ currents by NK2
receptors (Fig. 3) as well as
the inhibition of Ca2+ currents by NK3
receptors (Fig. 4) was reversed
by the protein kinase C inhibitor bisindolylmaleimide at concentrations that
had no direct effects (Fig.
2C) on basal Ca2+ currents either
before or after the administration of the inactive NK1 agonist,
[Sar9, Met11]-substance P
(Fig. 2). Further indirect
support for a role of protein kinase C in the neurokinin receptor signaling
pathway was produced by experiments with the phorbol ester, phorbol
12,13-dibutyrate, which both enhanced (at low concentrations) and inhibited
(at high concentrations) Ca2+ channel currents in dorsal
root ganglia neurons. These changes were also reversed by bisindolylmaleimide
(Fig. 6). The facilitatory
effect of phorbol 12,13-dibutyrate was not prevented by blocking L- or N-type
Ca2+ channels individually with appropriate antagonists,
indicating that both types of Ca2+ channels are
facilitated by activation of protein kinase C. Similarly, the effects of
-conotoxin GVIA or Bay K 8644 were unaffected by enhancement of
Ca2+ channels by either substance P or the
NK2 agonist. This suggests that both N- and L-type
Ca2+ channels may be enhanced by activation of
NK2. This conclusion is consistent with previous findings by Hall
et al. (1995), which suggested
that both N- and L-type Ca2+ currents are increased by a
constitutively active protein kinase C injected intracellularly in dorsal root
ganglia neurons. Protein kinase C has also been reported to increase L- and
N-type Ca2+ currents in central dopaminergic neurons
(Uramura et al. 2001). Other
types of Ca2+ channels presumably contribute very little
to the facilitatory effects of phorbol 12,13-dibutyrate because blockers of P-
and T-type Ca2+ channels did not alter the facilitation
in our experiments. Since phorbol 12,13 dibutyrate enhanced both N- and L-type
Ca2+ channels, it is reasonable to conclude that the
action of NK2 receptors to enhance L- and N-type
Ca2+ channels in dorsal root ganglia neurons by
activating protein kinase C occurs in a similar fashion.
High concentrations of phorbol 12,13-dibutyrate inhibited
Ca2+ currents in this study, although to a larger extent
than the NK3 agonist, [MePhe7]-neurokinin B. After
inhibition of Ca2+ currents by
[MePhe7]-neurokinin B, -conotoxin GVIA blocked a lesser
fraction of Ca2+ currents (36 vs. >50%), and Bay K
produced a larger increase in currents (34 vs. <25%) than before
inhibition, suggesting that N-type Ca2+ currents or a
fraction of -conotoxin GVIA-sensitive Ca2+
currents were more selectively inhibited by [MePhe7]-neurokinin B.
Indeed, there is ample evidence that NK2
(Catalioto et al. 1998;
Takeda et al. 1992) and
NK3 (Melcangio and Bowery 1999) receptors are coupled by a
pertussis-insensitive G protein to phospholipase C and protein kinase C
activation. The large inhibitory effects of high concentrations of phorbol
12,13 dibutyrate also suggest that, unlike [MePhe7]-neurokinin B or
substance P–mediated attenuation of facilitation, phorbol 12,13
dibutyrate may be reducing both L- and N-type Ca2+
channel activities in dorsal root ganglia neurons or may have direct,
nonspecific effects on Ca2+ channels above certain
concentrations.
Protein kinase C is activated by signal transduction mechanisms that
stimulate the break down of phospholipids by phospholipase C and/or D to
generate diacyl glycerol and IP3. The protein kinase C (PKC) family
consists of ≥12 different polypeptides
(Hug and Sarre 1993). Protein
kinase C subtypes can be divided in three major groups, according to their
primary structure and functional similarities: 1)
Ca2+-dependent or conventional (c), PKCc,
which needs both Ca2+ and diacylglycerol for activation;
2) Ca2+-independent or novel (n),
PKCn; and 3) atypical (a), PKCa, which
requires neither Ca2+ nor diacylglycerol for activation
(Way et al. 2000). Different
PKC isoforms may exhibit different substrate specificity and subcellular
distribution (Mochly-Rosen and Gordon
1998). One way to confer selectivity to such a diverse group of
isoenzymes is through specific anchoring proteins
(Csukai and Mochly-Rosen 1999;
Mochly-Rosen and Gordon 1998).
A question that would be purely speculative at this stage is whether the
facilitatory or inhibitory effects reported here would be mediated by
different protein kinase C subtypes with substrate specificity, selective
coupling of NK2 and NK3 receptors with different
subtypes of protein kinase C, or some other cellular localization mechanism.
This deserves further investigation. Indeed, the well-known upregulation of
L-type Ca2+ channels by chronic ethanol exposure is
mediated selectively by PKC
and the upregulation of N-type
Ca2+ channels is inhibited by a selective peptide
inhibitor of PKC
, although both of these effects are at the level of
gene regulation rather than phosphorylation of Ca2+
channel subtypes (McMahon et al.
2000). Use of selective PKC activators and inhibitors suggested
that PKC is the isoform responsible for inhibition of voltage-dependent
Ca2+ channels in adrenal chromaffin cells, although the
identity of Ca2+ channel subtype was not determined
(Sena et al. 2001). In neurons
from the major pelvic ganglion of rats, PKC activated by a selective
M1 agonist preferentially upregulated only the L-type
Ca2+ currents, although both N- and L-type
Ca2+ channels are expressed in these neurons
(Sculptoreanu et al.
2001).
Possible implication of neurokinin modulation of Ca2+ channels in the release of peptide neurotransmitters
Since both L- and N-type Ca2+ channels seem to be
involved in release of neuropeptides, including neurokinins
(Kageyama et al. 1997), and
the peptide release sites (large granular synaptic vesicles,
Otsuka and Yoshioka 1993) may
be distant to the conventional docked-small vesicle release sites, neurokinin
autoreceptors (Harding et al.
1999; Malcangio and Bowery
1999) would be expected to modulate their own release with PKC
activation as an intermediary in the process
(Barber and Vasko 1996;
Hingtgen and Vasko 1994) and
might modulate the release of other neurotransmitters
(Malcangio and Bowery 1999).
Thus the mechanism of Ca2+ channel modulation we
describe here may have important consequences in modulation of
neurotransmitter release.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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Address for reprint requests: A. Sculptoreanu, Univ. of Pittsburgh, School of Medicine, Dept. of Pharmacology, E1304 Biomedical Science Tower, Pittsburgh, PA 15261 (E-mail: ads5{at}pitt.edu).
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, 0.05 µM; 
, 0.2 µM), and SB
235,375 after SP (0.2 µM, 
) for the experiment shown in A.
C: effect of SP (0.2 µM, 2) and BIS after SP (0.5 µM,
3) in 1 dorsal root ganglia neuron. D: time course of
control (
