Prince of Wales Medical Research Institute, University of New South Wales, Randwick, New South Wales 2031, Australia
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ABSTRACT |
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Davies, Philip J.,
David R. Ireland,
Juan Martinez-Pinna, and
Elspeth M. McLachlan.
Electrophysiological Roles of L-Type Channels in Different
Classes of Guinea Pig Sympathetic Neuron.
J. Neurophysiol. 82: 818-828, 1999.
The electrophysiological
consequences of blocking Ca2+ entry through L-type
Ca2+ channels have been examined in phasic
(Ph), tonic (T), and
long-afterhyperpolarizing (LAH) neurons of intact guinea
pig sympathetic ganglia isolated in vitro. Block of Ca2+
entry with Co2+ or Cd2+ depolarized
T and LAH neurons, reduced action
potential (AP) amplitude in Ph and LAH
neurons, and increased AP half-width in Ph neurons. The
afterhyperpolarization (AHP) and underlying Ca2+-dependent
K+ conductances (gKCa1 and
gKCa2) were reduced markedly in all classes. Addition of
10 µM nifedipine increased input resistance in LAH neurons, raised AP threshold in Ph and
LAH neurons, and caused a small increase in AP
half-width in Ph neurons. AHP amplitude and the
amplitude and decay time constant of gKCa1 were reduced by nifedipine in all classes; the slower conductance,
gKCa2, which underlies the prolonged AHP in
LAH neurons, was reduced by 40%. Surprisingly, AHP
half-width was lengthened by nifedipine in a proportion of neurons in
all classes; despite this, neuron excitability was increased during a
maintained depolarization. Nifedipine's effects on AHP half-width were
not mimicked by 2 mM Cs+ or 2 mM anthracene-9-carboxylic
acid, a blocker of Cl channels, and it did not modify
transient outward currents of the A or D types. The effects of 100 µM
Ni2+ differed from those of nifedipine. Thus in
Ph neurons, Ca2+ entry through L-type
channels during a single action potential contributes to activation of
K+ conductances involved in both the AP and AHP, whereas in
T and LAH neurons, it acts only on
gKCa1 and gKCa2. These results differ from the results in rat superior cervical ganglion neurons, in which
L-type channels are selectively coupled to BK channels, and in
hippocampal neurons, in which L-type channels are selectively coupled
to SK channels. We conclude that the sources of Ca2+ for
activating the various Ca2+-activated K+
conductances are distinct in different types of neuron.
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INTRODUCTION |
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The somatic membranes of mammalian sympathetic
postganglionic neurons bear several types of voltage-dependent
Ca2+ channel that potentially are activated
during the action potential (AP). In freshly dissociated somata of rat
superior cervical ganglion (SCG), 80% of the current during action
potential waveforms passes through N-type, 5% through L-type, and 1%
through P-type channels; 14% of the current is resistant to blockade
by known antagonists (Toth and Miller 1995). Q-type
channels sensitive to
-conotoxin MVIIC (McDonough et al.
1996
) and low-voltage-activated T-type channels
(Schofield and Ikeda 1988
) have not been detected in rat
SCG neurons. However T-type channels recently have been demonstrated in
noradrenergic neurons of the rat pelvic ganglion (Zhu et al. 1995
), indicating that sympathetic neurons in different
locations can express distinct channel types. The populations of
Ca2+ channels expressed in sympathetic neurons of
other species have not been identified. Neither is it clear, even in
central neurons, whether the Ca2+ channels
expressed in somal membranes are representative of the entire
population present in intact neurons as channel distribution can differ
between soma and dendrites (Ahlijanian et al. 1990
).
Ca2+ influx during the AP has
electrophysiological consequences, particularly in sympathetic neurons,
by modifying AP configuration (Belluzzi and Sacchi 1991;
Davies et al. 1996
) and initiating a prolonged
afterhyperpolarization (AHP) (Cassell and McLachlan 1987b
). All rat SCG neurons have a similar electrophysiological phenotype, firing phasically at the beginning of a maintained depolarization (Davies et al. 1996
; Wang and
McKinnon 1995
). However, sympathetic neurons in the guinea pig
behave in three distinctive ways when depolarized: phasic
(Ph), tonic (T), and long afterhyperpolarizing (LAH) neurons have been distinguished (Keast et al.
1993
; McLachlan and Meckler 1989
). Major
features of these classes of neuron are two distinct
Ca2+-activated K+
conductances that underlie the AHP. Sympathetic neurons in all classes
have a conductance, gKCa1, which is activated rapidly and
decays with a time constant of 100-150 ms. LAH neurons
have, in addition, a second, slower
Ca2+-activated K+
conductance, gKCa2, that prolongs the AHP for several
seconds (Cassell and McLachlan 1987b
). This is largely
mediated by Ca2+-activated release of
Ca2+ from internal stores (CICR) (Jobling
et al. 1993
).
Voltage-dependent Ca2+ influx also modifies the
action potential itself. In rat SCG neurons (Davies et al.
1996), Ca2+ entering through L-type
Ca2+ channels activates large conductance
(BK type) Ca2+-dependent K+ channels
contributing to AP repolarization so that blockade of L-type channels with nifedipine prolongs AP half-width. In contrast, the AHP follows activation of small conductance (SK type)
Ca2+-dependent K+ channels
primarily by Ca2+ influx through N-type
channels. Ca2+ entering through
Ca2+ channels resistant to all toxins prolongs
the AHP in a proportion of rat SCG neurons by releasing
Ca2+ from intracellular stores. Thus in these
neurons, Ca2+ entry through particular types of
channel has selective actions. However, we recently have found that
N-type channels are involved in activation of both BK and SK channels
in Ph neurons in the guinea pig lumbar paravertebral chain
(Ireland et al. 1998
).
In the present study, we have compared the physiological functions of
Ca2+ entry in modifying the characteristics of
the AP and AHP in the three classes of guinea pig sympathetic ganglion
cell. We have focused on the effects of selective blockade of L-type
channels using nifedipine. The data indicate that the electrical
consequences of Ca2+ entry through L-type
channels are distinctive for each class of neuron. They confirm that
L-type channels in sympathetic neurons can admit quantities of
Ca2+ sufficient to modify the AP itself, as well
as the ensuing AHP, and that the coupling between
Ca2+ channels and
Ca2+-activated K+ channels
in many guinea pig neurons differs from that in rat SCG neurons.
Furthermore sympathetic neurons differ from hippocampal somata, in
which L-type channels are coupled selectively only to SK channels
(Marrion and Tavalin 1998).
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METHODS |
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Guinea pigs (150-300 g of either sex) were deeply anaesthetized
with pentobarbitone (80 mg/kg ip) and exsanguinated by perfusion through the descending thoracic aorta with oxygenated physiological salt solution. The SCG, lumbar paravertebral chain ganglia (LSC) between L1 and L6, coeliac
ganglion or the inferior mesenteric ganglion and attached nerve
branches were dissected and studied in different experiments. Ganglia
were pinned out in vitro and superfused with oxygenated physiological
salt solution at 35°C [composition (in mM): 151 Na+, 4.7 K+, 2.0 Ca2+, 1.2 Mg2+, 144.5 Cl, 1.3 H2PO4
, 16.3 HCO3
, and
7.8 glucose, pH 7.2-7.4]. These procedures were approved by the
Animal Care and Ethics Committee of the University of New South Wales.
Intracellular recordings were made using microelectrodes filled with
0.5 M KCl (resistance 70-150 M
) and records taken in bridge mode,
single-electrode current clamp (s.e.c.c), and single-electrode voltage
clamp (s.e.v.c) as described in detail previously (Cassell et
al. 1986
; Davies et al. 1996
).
Cells were classified as Ph, T, or
LAH, and their passive electrical properties were determined
as described previously (Cassell et al. 1986;
Keast et al. 1993
). Steady-state current-voltage relations from a holding potential of
60 mV were determined between AP threshold and values more negative than
90 mV near the end of a
250-ms hyperpolarizing current step. Passive electrical properties were
determined at a holding potential of
60 to
65 mV where the
current-voltage relationship was linear.
APs were generated after a brief (10-20 ms) depolarizing current step
from resting membrane potential (RMP); they were differentiated digitally and threshold voltage for initiation of the action potential determined from the point at which the voltage differential
(dV/dt) rose above 10 V/s. Because the RMP
differed among classes, differences in the voltage threshold and
amplitude of the AP and the amplitude of the AHP were compared using
the absolute level of membrane potential. Depolarizations produced by
drugs were compensated for by passing current to hold the potential at
values more negative than 50 mV for measurements of APs. Effects on
the repolarization phase were evaluated by integrating the area of the
negative component of the differential (dV/dt),
in some instances over only its latter part.
The time course of the AHP was defined by measuring its half-width (i.e., duration at half-peak amplitude). This measurement is very much dependent on the peak amplitude of the AHP: if the initial hyperpolarization is relatively large, the half-width may be brief even though there is a significant prolonged component (e.g., in LAH neurons). Unfortunately we found that other measures, such as integration of the voltage change during the AHP, were no more satisfactory than half-width as descriptors of the changes in AHP configuration. The time when the peak of the AHP occurred was measured from the point when the membrane repolarized to resting membrane potential until the peak of the afterpotential.
Outward tail currents were generated after a voltage command step
(20-50 ms) that elicited only one "action current" (Cassell and McLachlan 1987b). The time course of gKCa1 in
LAH neurons was derived by subtracting a function describing
the sum of two exponentials fitted to gKCa2, from the
overall tail current (see Cassell and McLachlan 1987b
).
The amplitude of gKCa1 was determined by extrapolating the
exponential fitted to its decay except in a few cases in which the peak
current fell below this value, when it was measured directly. The
amplitude of gKCa2 was measured directly from the peak
current. The maximum amplitude of the A-current (IA) was measured at the end of a
250-ms voltage command step from
100 mV to a holding potential of
40 mV. Activation and inactivation of
IA and
ID were examined using standard
protocols described previously (Cassell et al. 1986
;
Inokuchi et al. 1997
).
RMP was measured as the difference between the potentials immediately before and after withdrawal of the microelectrode.
Drugs used
Nifedipine and anthracene-9-carboxylic acid (9AC) were obtained
from Sigma (Castle Hill, NSW, Australia). Both were dissolved initially
in ethanol and then diluted in physiological solution to reach the
final working concentration. Care was taken when using nifedipine to
minimize its exposure to light. Solutions containing drugs were added
to the bath by transferring the inlet of the perfusion system to a
solution containing the stated concentration. Effects of all drugs were
recorded 15 min after this when a steady state of block had been
achieved (see Davies et al. 1996
). The effects of 9AC
did not change after only 5-min exposure, although records were taken
10-20 min later. We have assumed in this study that addition of 10 µM nifedipine provides block of L-type Ca2+
channels. Unspecific blocking actions by nifedipine on
Na+ or K+ channels are
unlikely as there were no detectable changes in the amplitude of the AP
overshoot that might reflect blockade of voltage-dependent
Na+ channels and in T and LAH neurons nifedipine
had no effect on AP repolarization, suggesting it did not directly
block BK, delayed rectifier or A-type channels involved in AP
repolarization (Inokuchi et al. 1997
).
Statistical analysis
All values are expressed as means ± SE. All recorded
parameters were tested for equality of variance between groups
(F-ratio and Bartlett's test, = 0.05) and then
tested using appropriate parametric or nonparametric tests. Differences
between properties in control and drug solutions were tested using a
paired t-test or Wilcoxon signed-rank test. The effect of
Ca2+ blockade or addition of nifedipine was
compared between classes on the ratios of drug/control values using an
ANOVA and multivariate ANOVA to determine any difference on the basis
of all tests applied in the program, Superanova (Abacus Software,
Berkeley, CA). Differences between groups were tested using an unpaired
t-test or Mann-Whitney test. Differences in proportions of a
particular trait among the three classes were tested using a
2 test, and the adjusted residuals were
calculated to identify which classes were responsible for the
significant overall
2 value. All reported
significant differences had P values <0.05.
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RESULTS |
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Electrophysiological properties of three classes of sympathetic neuron
RMP and passive electrical properties (Tables
1A and
2A) were similar to those
previously reported (Cassell et al. 1986; Keast
et al. 1993
). The characteristics of the action potential and
afterhyperpolarization are detailed in Tables 1, B and
C, and 2, B and C.
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Effect of blockade of Ca2+ entry
PASSIVE ELECTRICAL PROPERTIES. Ca2+ entry was blocked by replacement with Co2+ (n = 25), or addition of the nonspecific Ca2+ channel blocker, Cd2+ (300 µM; n = 8). There was significant membrane depolarization only in T and LAH neurons (Table 1A).
ACTION POTENTIAL.
Overall, blockade of Ca2+ influx produced a
significant decrease in the current required to reach threshold
(control 0.38 ± 0.04 nA;
Co2+/Cd2+ 0.31 ± 0.03 nA; n = 33; P = 0.01) but the absolute
threshold voltage at which the AP was initiated was unchanged (control
27 ± 2 mV;
Co2+/Cd2+
28 ± 1 mV; n = 33; P = 0.67). However, in
Ph and LAH neurons, both the overshoot of the AP
and max dV/dt decreased (Fig.
1, Table 1B), presumably
reflecting a reduction in total inward current.
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AFTERHYPERPOLARIZATION. Blockade of Ca2+ entry markedly reduced the AHP amplitude (control 12 ± 1 mV; Co2+/Cd2+ 6 ± 1 mV; n = 33; P < 0.0001) and its half-width (control 111 ± 12 ms; Co2+/Cd2+ 39 ± 6 ms; n = 33; P < 0.0001). While the peak of AHP occurred earlier, this was not significant (time to peak AHP, control 17 ± 3 ms; zero Ca2+ 10 ± 2 ms; n = 23; P = 0.06). Under voltage clamp, peak amplitude of gKCa1 and its decay time constant were reduced. These changes were present in all classes although the reduction in the decay time constant of gKCa1 was not significant in T neurons (Fig. 1, Table 1C). In LAH neurons, the long AHP and the slower component of the outward current, gKCa2, were virtually abolished by blockade of Ca2+ influx.
The effects of Ca2+ channel blockade were not different between the three classes of neuron (MANOVA, P = 0.60).FIRING PROPERTIES.
Following replacement of Ca2+ with
Co2+, the neurons became more excitable and fired
a greater number of APs during a prolonged depolarizing current step.
In Ph and LAH neurons, current steps that
elicited a single AP in control solution fired multiple APs following
Ca2+ blockade, whereas in T neurons, a given
level of suprathreshold depolarizing current elicited repetitive firing
at a higher frequency (see Fig. 8 in Davies et al. 1996).
Effect of blockade of L-type Ca2+ channels with 10 µM nifedipine
PASSIVE ELECTRICAL PROPERTIES. Addition of 10 µM nifedipine had no effect on passive membrane properties in Ph and T neurons whereas nifedipine caused a small increase in Rin in LAH neurons (Table 2A) which also depolarized slightly (P = 0.06). Current-voltage relations were not affected by nifedipine.
ACTION POTENTIAL. Nifedipine had no effect on the amplitude of the AP but threshold voltage was raised by 3-4 mV in Ph and LAH neurons (Table 2B). Max dV/dt was slightly reduced overall (control 224 ± 5 V/s; nifedipine 217 ± 5 V/s; n = 49; P = 0.02) (Fig. 1), although not significantly within any class (Table 2B). AP half-width was prolonged only in Ph neurons (by 4 ± 1%, Table 2B). The AP in T neurons was unaffected by nifedipine (Table 2B).
AFTERHYPERPOLARIZATION. Nifedipine reduced both AHP amplitude (control 12 ± 1; nifedipine 11 ± 1; n = 50; P = 0.0003) and the amplitude of gKCa1 (control 120 ± 10 pA; nifedipine 100 ± 10 pA; n = 47; P = 0.0001). In addition, in a subgroup of neurons with a relatively early AHP peak in control solution (<15 ms), nifedipine delayed the peak of the AHP (control 5 ± 1 ms; nifedipine 16 ± 3 ms; n = 26; P = 0.0005). In the remaining neurons with a slower onset AHP, nifedipine did not change the time at which the AHP peak occurred (control 45 ± 4 ms; nifedipine 43 ± 4 ms; n = 23; P = 0.5). The decay time constant of gKCa1 was briefer in nifedipine. These effects occurred to various extents in the different classes of neuron (Table 2C, Fig. 3).
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FIRING PROPERTIES. Addition of nifedipine had little effect on the firing properties during a just threshold depolarizing current step in any class of neuron, despite its actions in raising threshold. However in all neuron classes, the number of APs during larger current steps was usually greater than in control solution (Fig. 5). Thus the number of APs generated using a current step twice that necessary to trigger an AP was increased in all classes (Ph control 2.7 ± 0.6; nifedipine 4.2 ± 1.1; n = 12; P = 0.04; T control 3.1 ± 0.6; nifedipine 3.3 ± 0.5; n = 14; P = 0.04; LAH control 1.7 ± 0.3; nifedipine 2.8 ± 0.7; n = 9; P = 0.02).
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Contribution of other Ca2+-dependent channels to the AHP
The widening of the AHP by nifedipine, despite a reduction in gKCa1 (Table 2C), might be mediated by block of Ca2+ entry through channels that do not provide Ca2+ for activation of gKCa1. Two possible conductances that might normally attenuate the AHP were examined:
(I) EFFECTS OF BLOCKADE OF T-TYPE
Ca2+ CHANNELS.
T-type Ca2+ channels are blocked by 10 µM
nifedipine (Randall and Tsien 1997). Activation of
T-type Ca2+ channels during AHP repolarization
might attenuate its time course by introducing an inward current. We
therefore tested the effects of 100 µM Ni2+, a
blocker of T-type Ca2+ channels (Fox et
al. 1987
). Addition of Ni2+ depolarized
T neurons (control
62 ± 2 mV;
Ni2+
60 ± 1 mV, n = 14;
P = 0.01) without significantly changing Rin. There was a small increase in AP
half-width which was significant only in T neurons
(Ph: control 1.34 ± 0.08 ms;
Ni2+ 1.38 ± 0.09 ms, n = 14; T: control 1.52 ± 0.10 ms;
Ni2+ 1.62 ± 0.09 ms, n = 13, P = 0.01; LAH: control
1.38 ± 0.14 ms; Ni2+ 1.40 ± 0.15 ms,
n = 6), but there were no significant effects on the
AHP or gKCa1 (n = 33). Addition of 10 µM nifedipine
in the presence of 100 µM Ni2+ further
increased AP half-width (control 1.23 ± 0.05 ms:
Ni2+ 1.30 ± 0.05 ms; nifedipine 1.45 ± 0.09 ms, n = 12, P = 0.02). These
data suggest that nifedipine's actions were not mediated by blockade
of T-type channels.
(II) EFFECTS OF BLOCKADE OF Ca2+-ACTIVATED
CHLORIDE CHANNELS.
In mouse SCG neurons, Ca2+ influx during the AP
activates a Cl current producing an
afterdepolarization (ADP) (De Castro et al. 1997
) which
attenuates the AHP. This ADP is blocked selectively by nifedipine,
which does not affect gKCa1 after a single action potential
(Martinez-Pinna et al. 1998
). Application of the
Cl
channel blocker, 9AC (2 mM), had no effect
on passive electrical properties or the AHP of 8 prevertebral
T neurons (see also Clark et al. 1998
). Neither did it
affect the amplitude of gKCa1, although it decreased its decay time
constant (control 118 ± 13 ms; 9AC 85 ± 9 ms;
n = 6; P = 0.03). In one T
neuron, in which 9AC had no effect on the AHP, addition of nifedipine
subsequently widened it. These observations suggest that the actions of
nifedipine on the AHP cannot be attributed to blockade of a
Ca2+-dependent Cl
conductance.
Contribution of voltage-dependent K+ currents to the AHP
The effect of nifedipine on AHP half-width is therefore most
easily explained if it modulates a voltage-dependent outward conductance activated during the AHP. We tested whether nifedipine had
effects on either the A-current (IA) which
is particularly prominent in T neurons (Cassell et
al. 1986), or the D-current, a slower transient
outward current, which is also present in some T neurons
(Inokuchi et al. 1997
). However,
IA amplitude, time course and
activation/inactivation characteristics were similar in control
solution and in nifedipine (n = 8 T
neurons). D-type currents which followed
IA in 7 T neurons were slightly
reduced in amplitude (control 0.15 ± 0.03 nA, nifedipine
0.09 ± 0.01 nA; P = 0.06) and abbreviated (decay
time constant: control 416 ± 54 ms; nifedipine 355 ± 56 ms;
P = 0.04) in nifedipine, but activation and inactivation
were unaffected. In addition, the presence of ID was not correlated with widening of the
AHP in nifedipine (
2 test, P = 0.93).
A time-dependent hyperpolarization-activated cationic conductance
(IH) attenuates the AHP in some central
neurons (Schwindt et al. 1988). Addition of 2 mM
Cs+ had no effect on the shape or size of the AHP
or gKCa1 in 3 T neurons. Further an effect on anomalous
rectification is unlikely as the membrane potential during the peak of
the AHP (see Tables 1 and 2) did not reach potentials at which
significant rectification occurs. There was no correlation between the
presence of inward rectification negative of
90 mV and the effect of
nifedipine on the AHP (
2 test, P = 0.38).
Differences in the effects of nifedipine between ganglia
Ph neurons comprise the only class which is represented in different ganglia in sufficient numbers to test the possibility that the effects of nifedipine differ between anatomic locations. Among Ph neurons, nifedipine caused an increase in Rin(+31 ± 8%) in prevertebral neurons (n = 5, P = 0.03) that did not occur in paravertebral neurons (Rin +1 ± 7%, n = 15); this change was significantly different between the groups (P = 0.01). The change in AP half-width was similar in paravertebral (+4.2 ± 1.1%) and prevertebral (+4.9 ± 2.1%) neurons. In contrast, the amplitude of gKCa1 was significantly reduced by nifedipine in prevertebral (gKCa1 -34 ± 12%; n = 5; P < 0.05) but not in paravertebral neurons (gKCa1 -6 ± 5%, n = 15); these changes were significantly different (P < 0.02). Despite the larger reduction in gKCa1, widening of the AHP was generally greater in prevertebral Ph neurons (+191 ± 122%, n = 5; cf. T neurons, +25 ± 8%, n = 18) than in paravertebral ones (+10 ± 7%, n = 15, P = 0.02).
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DISCUSSION |
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In this study, Ca2+ entry during the action
potential has been shown to have distinct effects on the
electrophysiological properties of each of the three main classes
(Ph, T, LAH) of neuron present in guinea pig
sympathetic ganglia. The new data on the consequences of
Ca2+ entry provide confirmatory evidence for
at least three main phenotypes of sympathetic neuron. In sympathetic
neurons, voltage-dependent Ca2+ influx is
responsible not only for the long AHP but, in Ph and LAH neurons, also determines the amplitude and
time course of the AP itself. Both roles of Ca2+
are partly mediated by its influx through L-type
Ca2+ channels, as revealed after selective
blockade with nifedipine. In addition, nifedipine had two actions that
were not seen when Ca2+ entry was blocked
nonspecifically, namely, to raise the threshold for action potential
initiation in Ph and LAH neurons and
to widen the half-width of the AHP in some neurons. Overall, the
results contrast with those of similar experiments on Ph
neurons in the rat SCG in which Ca2+ influx
through L-type channels led selectively to activation of BK-type
channels during AP repolarization (Davies et al. 1996). However it should be noted that the effects of nifedipine in
paravertebral Ph neurons were generally similar in both species.
The effects of Ca2+ entry through L-type
channels on the configuration of the AP and the afterpotentials in
sympathetic neurons (see also Martinez-Pinna et al. 1998) contrast with
the lack of effect in postganglionic (Callister et al.
1997
) and preganglionic parasympathetic neurons (Sah
1996
) as well as in central neurons (e.g., Pineda et al. 1998
)
where influx of Ca2+ through L-type channels
constitutes a significant proportion of the total
Ca2+ current (Ishibashi et al.
1995
). Following repetitive firing in hippocampal neurons, the
AHP (Moyer et al. 1992
; Norris et al.
1998
) and the underlying slow
Ca2+-dependent K+
conductance (Tanabe et al. 1998
) are blocked by L-type
channel antagonists although the APs themselves are unaltered.
Recently, in patches of somatic membrane of hippocampal neurons,
Ca2+ influx through L-type channels was shown to
activate only SK -type channels despite BK-type channels being present
within the same patch (Marrion and Tavalin 1998
). This
indicates that the sources of Ca2+ for activation
of Ca2+-sensitive channels are distinct in
different neurons and that it is unlikely that diffusion is necessarily
the determining factor.
Passive electrical properties
Blocking entry of extracellular Ca2+
depolarized the neurons by several mV (see also McLachlan 1977) but, in
T neurons, this was not accompanied by a change in
Rin, so that the effect probably did not
involve a decrease in a resting Ca2+-dependent
K+ conductance. However, depolarization of
LAH neurons was accompanied by an increase in
Rin. One explanation might be that gKCa2,
which is activated by Ca2+ influx through L-type
channels, is partly activated near RMP (see Tokimasa and Akasu 1995
).
The lack of effect of Cd2+, however, indicates
that other Ca2+-sensitive mechanisms have
opposing effects on resting conductance. As A-channels make an
important contribution to RMP in T neurons (see Cassell et
al. 1986
; Inokuchi et al. 1997
), it seems likely that resting membrane
conductance is regulated by distinct mechanisms in each neuron class.
Action potentials
Several features of the AP were shown to be modified selectively by Ca2+ blockade:
1) Although threshold for AP initiation was not affected
by Co2+ or Cd2+, it
rose by 2-3 mV in Ph and LAH neurons in nifedipine. A
similar effect of nifedipine has been described in guinea pig
hippocampal neurons (Higashi et al. 1990). This change
is difficult to explain unless inward current through
nifedipine-sensitive channels makes a significant contribution early in
the regenerative response. Blockade of N-type channels with
-conotoxin GVIA did not affect threshold in Ph neurons
(Ireland et al. 1998
).
2) The AP in T neurons was surprisingly
unaffected by blockade of Ca2+ entry, compared
with the effect on AP configuration in Ph and LAH neurons (see also Belluzzi and Sacchi 1991;
Davies et al. 1996
). In sympathetic neurons, at least three types of
K+ channels are activated during repolarization:
delayed rectifier channels, A-channels (Belluzzi and Sacchi
1991
; Inokuchi et al. 1997
) and BK-type channels
(Davies et al. 1996
; Ireland et al. 1998
). When Ca2+ entry was blocked, the
inflection during the repolarization phase disappeared from all classes
of neuron (as reported for other autonomic neurons, Callister et al.
1997
; Mochida and Kobayashi 1986
; Sah and McLachlan 1992
; Yoshimura et
al. 1986
). This inflection is determined by the net addition of inward
Ca2+ current and outward K+
current so that block of Ca2+ entry has complex
effects on repolarization. While the AP was widened by
Co2+/Cd2+ only in
Ph neurons (by ~16%), only a small component (25%) of this change occurred via L-type channels. In Ph neurons,
N-type channels play a much greater role in determining AP half-width but have no significant effects on amplitude and
dV/dt (Ireland et al. 1998
),
implying that Ca2+ entry through channels other
than N- or L-type must contribute. The failure of blockade of
Ca2+ entry to affect AP half-width in
T and LAH neurons suggests that these cells lack
BK channels.
Slow afterhyperpolarizations
A major effect of blocking Ca2+ entry was to
abolish the later phase of the AHP by inhibition of gKCa1 and gKCa2.
The channels underlying gKCa1 are largely apamin-sensitive SK channels
(Davies et al. 1996; Ireland et al. 1998
;
Jobling et al. 1993
). In Ph neurons of the
rat SCG (Davies et al. 1996
), as well as in other autonomic (Callister et al. 1997
; Sah
1996
) and many central neurons (Pineda et al.
1992
), most Ca2+ that activates SK
channels enters via N-type Ca2+ channels. Of
gKCa1, 60% depends on the Ca2+ provided via
N-type channels in guinea pig paravertebral Ph neurons (Ireland et al. 1998
); the present results indicate that
~15% of the rest comes through L-type channels (Tables 1 and 2) so that other Ca2+ channels must also contribute. In
contrast, ~40% of gKCa2 depends on Ca2+ entry
through L-type channels. This kinetically slow conductance is activated
via CICR from intracellular stores (Jobling et al. 1993
). The large effect of nifedipine on gKCa2 indicates that Ca2+ influx through L-type channels is important
for activation of CICR (Li and Hatton 1997
;
Osmanovic and Shefner 1993
).
Distribution of Ca2+ channels in sympathetic neurons
The effects of L-channel block on gKCa1 and gKCa2 are
relatively large if it is assumed that the proportions of channels
activated during the action potential in guinea pig neurons are the
same as those in rat SCG somata, in which only 5% of the
Ca2+ current is carried by L-type channels
(Toth and Miller 1995). Perhaps the
Ca2+ entering through L-type channels is much
more effective in activating K+ channels than the
Ca2+ that enters through the more numerous N-type
channels. This might result from the closer juxtaposition of L-type
channels than N-type channels to the relevant K+
channels. This explanation is hard to reconcile with the apparently rapid activation of gKCa1 by Ca2+ from both
sources. Another explanation might be that the majority of L-type
channels are located on dendrites and would not contribute to the
current measured in dissociated somata. If this were the case, all
types of K+ channel that are activated by
Ca2+ from L-type channels are probably also
distributed over the dendrites. In this respect, in mouse SCG neurons,
in which Ca2+-activated
Cl
channels are thought to be located on distal
dendrites (De Castro et al. 1997
), nifedipine blocks
their activation without affecting the SK channels underlying the AHP
(Martinez-Pinna et al. 1998
). In this context, it is
interesting that L-type channel blockade had no effects on
parasympathetic neurons which have few or very small dendrites
(Callister et al. 1997
).
Unexpected effects of nifedipine
Although nifedipine reduced both
Ca2+-activated K+
conductances in most sympathetic neurons, it increased AHP half-width
and generated a secondary "belly" in a subgroup, particularly
prevertebral neurons. Such an effect did not occur when all
Ca2+ influx was blocked. Nifedipine probably
interfered with the regulation of a voltage-dependent conductance in
all classes of neuron as the decrease in gKCa1 it produced was very
rarely accompanied by a reduction in the AHP. We found no evidence for
involvement of T-channels, Ca2+-activated
Cl channels or voltage-dependent
K+ channels of the A-, D- or H-types. Two other
voltage-dependent K+ currents might conceivably
contribute to abbreviation of the AHP under normal conditions, namely,
the time-independent inward rectifier, IIR
(Wang and McKinnon 1996
) and the M-current
(Selyanko and Brown 1996
). Unfortunately we were unable
to test these possibilities directly, as the appropriate antagonists,
Ba2+ and muscarinic agonists, are not specific
and also inhibit gKCa1 (Cassell and McLachlan 1987a
).
The consequences of L-type channel block in increasing excitability
(Fig. 5) imply that the reduction in gKCa1 is more important
functionally than AHP prolongation.
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CONCLUSIONS |
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The results confirm that Ca2+ influx
during the AP markedly affects the excitability of guinea pig
sympathetic neurons of all classes, mainly by its effects on the slow
Ca2+-activated K+
conductances (gKCa1 and gKCa2) that underlie the AHP. In this respect,
the role of L-type channels differs from its roles in rat and mouse SCG
neurons. Further, in Ph neurons of rats and guinea pigs,
unlike other neurons so far studied, L-type Ca2+
channels are activated during a single action potential, providing Ca2+ that modifies repolarization through
activation of BK channels. Thus Ca2+
entering through L-type channels activates BK, SK and possibly other
K+ channels in guinea pig sympathetic neurons
(see also Wisgirda and Dryer 1994). The same diverse targets are
modulated by the Ca2+ that enters through N-type
channels (Ireland et al. 1998
).
The present findings contrast with three examples of type-specific
functional linkages: 1) between L-type channels and BK channels, and between N-type channels and SK channels, in rat SCG cells
(Davies et al. 1996; see also Wisgirda and Dryer
1994
), 2) between L-type channels and SK channels,
and N-type channels and BK channels, in hippocampal neurons
(Marrion and Tavalin 1998
) and 3) between
L-type channels and Cl
channels, and again
between N-type channels and SK channels, in mouse SCG cells
(Martinez-Pinna et al. 1998
). Our observations are not
evidence against the concept that there is a close physical association
between a voltage-dependent Ca2+ channel and the
Ca2+-sensitive channel(s) it activates. It might
simply be that the pairing is not always specified by channel type. We
conclude that the linkages are distinct in different types of neuron
and hypothesize that associated pairs of channels may have specific
distributions in different parts of the cell.
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ACKNOWLEDGMENTS |
---|
We thank P. Lund for technical assistance, J. Jamieson and Dr. Martin Stebbing for computer programming assistance, and Dr. Ross Odell for statistical advice.
This work was supported by the National Health and Medical Research Council of Australia. D. R. Ireland holds an Australian Postgraduate Award. J. Martinez-Pinna's travel was supported by funds from the Dirección General de Enseñanza Superior, Spain.
Present address of J. Martinez-Pinna: Instituto de Neurociencias, Universidad Miguel Hernández, Alicante, Spain.
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FOOTNOTES |
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Address for reprint requests: P. Davies, Prince of Wales Medical Research Institute, High St., Randwick, NSW 2031, Australia.
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 31 December 1999; accepted in final form 26 March 1999.
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REFERENCES |
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