Department of Neurophysiology, Research Institute Neurosciences, Vrije Universiteit, 1081 HV Amsterdam, The Netherlands
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ABSTRACT |
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Van Soest, Paul F.,
Johannes
C. Lodder, and
Karel S. Kits.
Activation of Protein Kinase C by Oxytocin-Related Conopressin
Underlies Pacemaker Current in Lymnaea Central
Neurons.
J. Neurophysiol. 84: 2541-2551, 2000.
The vasopressin/oxytocin-related neuropeptide
Lys-conopressin activates two pacemaker currents in central neurons of
the mollusk Lymnaea stagnalis. A high-voltage-activated
current (I-HVA) is activated at potentials greater than 40
mV and resembles pacemaker currents found in many molluscan neurons. A
low-voltage-activated current (I-LVA) activates throughout
the range of
90 to 0 mV. Based on sequence homologies,
Lymnaea conopressin receptors are thought to couple to
Q-type G proteins and protein kinase C (PKC). Alternatively,
agonist-induced pacemaker currents in molluscan neurons have
traditionally been attributed to cAMP-dependent protein kinase (PKA)
activation. Accordingly, this study aimed at resolving possible
involvement of cAMP/PKA and diacylglycerol/PKC in the conopressin
response. Injection of cAMP into anterior lobe neurons induced a slow
inward current with a voltage dependence resembling that of
ILVA (and not
IHVA). However, lack of effect of the
phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine and the absence
of cross-desensitization between cAMP and conopressin suggest that
neither current is dependent on intracellular cAMP. The PKC-activating
phorbol ester 12-O-tetradecanoylphorbol 13-acetate (but not
inactive phorbol 12-myristate 13-acetate) mimicked activation of
IHVA, but not
ILVA, and occluded subsequent responses to conopressin. Activation of
IHVA was blocked by general protein
kinase inhibitors and the PKC-inhibitor GF-109203X. Modulation of the
calcium buffering capacity of the pipette medium did not affect the
conopressin response, suggesting that calcium dynamics are not of major
importance. We conclude that conopressin activates the ion channels
carrying ILVA and
IHVA through different
second-messenger cascades and that PKC-dependent phosphorylation
underlies activation of IHVA.
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INTRODUCTION |
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The molluscan
neuropeptide Lys-conopressin, a vasopressin/oxytocin analogue, is
expressed abundantly by neurons in the anterior lobe of the right
cerebral ganglion of Lymnaea stagnalis (Van Kesteren
et al. 1995a). Most of these neurons also express a G protein-coupled conopressin receptor. So far, two receptor subtypes have been identified, both of which are related to mammalian
vasopressin V1 and oxytocin receptors (Van Kesteren et al.
1995b
, 1996
). The abundance of both conopressin
and its receptors makes the anterior lobe of Lymnaea an
advantageous system to study the effects of this neuropeptide.
Previously, we have studied the responses of isolated anterior
lobe neurons to conopressin application under voltage-clamp conditions
(Van Soest and Kits 1997, 1998
). In most
of the anterior lobe neurons, conopressin activates either one or both
of two distinct persistent inward currents. The first type, a highly voltage-dependent current that is activated at potentials above
40 mV
(IHVA), occurs in most of the cells.
The second type, a weakly voltage-dependent current that is activated
at all potentials between
90 and +10 mV
(ILVA), occurs in a minority of
neurons, and only in combination with
IHVA. Both currents are carried mainly by sodium ions.
The HVA and LVA currents differ in some important aspects.
Desensitization experiments revealed that the amplitude of the LVA
current decreases strongly within a few minutes in the continuous presence of the peptide, while the HVA current showed hardly any desensitization. Similarly, the LVA current declined rapidly after wash
out of conopressin, while the HVA current takes several minutes to
disappear. Thus a single peptide can modulate various physiological parameters at different time scales. The observations suggested that
different signal transduction mechanisms, and possibly different conopressin receptors underlie activation of the HVA and LVA currents (Van Soest and Kits 1997).
So far, the identity of the second-messenger systems involved in the conopressin responses has remained unknown. Insight in this matter will shed light on the complex mechanisms through which conopressin controls the activity of cells by differentially modulating various ion channels. This seems especially interesting in the case of the HVA current, whose activation substantially outlasts the actual conopressin application.
The responses of molluscan neurons to vasopressin, oxytocin,
and related peptides have previously been attributed to increases in
intracellular cAMP. A vasopressin-related peptide factor extracted from
molluscan ganglia enhanced bursting pacemaker activity in an identified
Otala neuron (Ifshin et al. 1975). A peptide
extract with similar properties was shown to increase levels of cAMP in Helix and Aplysia neurons, and the burst-inducing
effects of these peptides were mimicked by application of cAMP and
treatment with the phosphodiesterase-inhibitor
3-isobutyl-1-methylxanthine (IBMX) (Levitan et al. 1979
;
Treistman and Levitan 1976
). Similarly, the persistent
sodium current induced by oxytocin in an identified Achatina
neuron was mimicked by injection of cAMP, augmented by IBMX, and
partially blocked by cAMP-dependent protein kinase (PKA) inhibitors, indicating that the response is mediated by
PKA-dependent protein phosphorylation (Funase
1990
).
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Moreover, cAMP has been shown to induce or to mediate agonist-induced
activation of slow sodium currents in many molluscan preparations (see,
e.g., Aldenhoff et al. 1983; Connor and
Hockberger 1984
; Deterre et al. 1981
), and to
enhance the opening frequency of single channels carrying inward
current in Pleurobranchaea neurons (Green and
Gillette 1983
). Although many reports indicate that cAMP exerts
its action on ion channels through PKA-mediated phosphorylation, a
sodium channel that is directly gated by cAMP was identified in
Pleurobranchaea neurons (Sudlow et al. 1993
). In Lymnaea, intracellular injection of cAMP induces a slow
sodium current in neurons involved in feeding behavior (McCrohan
and Gillette 1988
). Thus it seems reasonable to propose that a
cAMP-dependent pathway might underlie the conopressin-induced
persistent sodium LVA and HVA currents.
On the other hand, the homology of the Lymnaea conopressin
receptors to vertebrate vasopressin V1 and oxytocin receptors, both of
which interact with G proteins of the Q type, suggests that they are
likely to couple to phospholipase C (Van Kesteren et al.
1995b, 1996
). Thus conopressin might activate
protein kinase C (PKC) through production of 1,2-diacylglycerol (DAG)
(for reviews see Nishizuka 1984
, 1995
).
Further support for this scheme was supplied by in vitro reconstitution
of the conopressin receptors. When expressed in Xenopus
oocytes, both types of Lymnaea conopressin receptor could
activate a calcium-dependent anion channel, probably through
G
q-mediated calcium
release from intracellular stores (Van Kesteren et al.
1995b
, 1996
).
Various studies have suggested a role for PKC in molluscan neurons. An
endogenous PKC was identified in the CNS of Aplysia (de Riemer et al. 1985a), and PKC activity was
subsequently characterized in further studies (e.g.
Pepio et al. 1998
; Sossin and Schwartz 1994
; Sossin et al. 1993
). Activation of PKC was
shown to stimulate high-voltage-activated calcium currents in
neuroendocrine cells in Aplysia (de Riemer et al.
1985b
) and in Lymnaea (Dreijer and Kits
1995
). Similarly, a number of papers addressed PKC modulation of potassium currents (e.g. Critz and Byrne 1992
;
Sugita et al. 1994
, 1997
). However, there
is little evidence for the activation of slow inward currents by PKC. A
notable exception is found in the bag cells of Aplysia,
where a nonselective cation channel is up-regulated by PKC and
presumably underlies the prolonged firing pattern, known as
afterdischarge (Wilson et al. 1996
,
1998
). Treatment with a phorbol ester or a DAG analogue
induced a slow and weakly voltage-dependent inward current in
Aplysia motor neurons, but this current was not further
characterized (Sawada et al. 1989
).
Thus the present study was undertaken to elucidate the signal transduction pathway underlying activation of the conopressin induced currents, focusing on the possible involvement of cAMP-activated PKA and DAG-activated PKC.
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METHODS |
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Animals and preparations
All experiments were performed on isolated anterior lobe neurons from adult, laboratory-bred specimens of the pond snail Lymnaea stagnalis (L.). The animals were kept under a 12:12-h light:dark regime in aerated, circulating water at a temperature of 20°C and were fed lettuce ad libitum. To obtain isolated neurons, the CNS was dissected out and incubated in an 0.2% solution of trypsin (Sigma type III) in HEPES-buffered saline (HBS) at 37°C for 35 min. After the incubation period, the CNS was rinsed in HBS and pinned down in a dish. The outer layers of connective tissue covering the anterior lobe of the right cerebral ganglion were removed, and the anterior lobe was severed from the CNS. Single anterior lobes were transferred to 35-mm plastic culture dishes (Corning Costar, Cambridge, MA) and mechanically dissociated. After dissociation, the cells were allowed to sit for at least 30 min before the dish was transferred to the experimental setup.
Recording technique
Whole cell voltage-clamp recording of isolated neurons was
performed in a standard patch-clamp setup, containing an Axopatch 1C or
200B (Axon Instruments, Burlingame, CA) patch-clamp amplifier. Current
and voltage traces were digitized using CED 1401 (Cambridge Electronic
Design, Cambridge, UK) or Digidata 1200 (Axon Instruments) AD/DA
convertors and recorded on an IBM-compatible PC using custom software
(developed by P. F. van Soest) or p-Clamp (Axon Instruments). Large-tip patch pipettes were pulled from borosilicate capillaries (Clark Electromedical Instruments, Reading, UK). Series resistance normally amounted to 1-3 M, approximately 75% of which could be
compensated for.
Pseudo steady-state current-voltage (I-V) relations were
obtained by applying voltage ramp protocols in which the command potential was swept from 90 to +10 mV at a rate of 5 mV/s. Ramps were
generally applied at 2-min intervals. Conopressin-induced current
profiles were obtained by subtracting the control I-V relation from that obtained in the presence of conopressin. Data are
presented as means ± SD, and statistical significance is
indicated by P-values obtained from Student's
t-tests, unless otherwise stated.
Drugs and solutions
Standard HBS was composed of (in mM) 30 NaCl, 1.7 KCl, 10 NaCH3SO4, 1.5 MgCl2, 4 CaCl2, 5 NaHCO3, and 10 HEPES, pH set at 7.8 with NaOH.
The standard pipette solution consisted of (in mM) 29 KCl, 2 NaCl, 10 HEPES, 11 ethylene glycol-bis (-aminoethyl ether)-N,N,N',N'-tetraacetic
acid (EGTA), 2.3 CaCl2, 2 Mg-ATP, and 0.1 GTP-Tris, pH set at 7.4 with KOH (~35 mM). The calculated concentration of free Ca2+ in the pipette medium
was 10 nM (cf. Stockbridge 1987
). Cesium pipette medium
had the same composition, except for the KCl, which was replaced by 29 mM of CsCl, and had its pH set with CsOH.
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) pipette medium was identical to cesium pipette medium except for EGTA, which was replaced by 11 mM BAPTA cesium salt.
Adenosine 3',5'-cyclic monophosphate (cAMP) was dissolved in water supplemented with an equimolar amount of KOH, 50 mM KCl, and 10 mM HEPES, buffered at pH ~7.4. Stock solutions ranging from 1 to 20 mM were injected intracellularly by means of pressure ejection from blunt microelectrodes. Injection pressure and duration were adjusted to induce stable responses as required in most experiments. To obtain desensitizing responses (in the cross-desensitization experiments) cAMP was injected at 20 mM.
The PKC-activating phorbol ester 12-O-tetradecanoylphorbol
13-acetate (TPA, also known as phorbol 12-myristate 13-acetate or PMA)
and the inactive phorbol ester 4--phorbol 12-myristate 13-acetate
(4-
-PMA) were obtained from Research Biochemicals International
(Natick, MA). The phorbol esters were dissolved in dimethylsulfoxide
(DMSO) and diluted to a final concentration of 1 µM in HBS (final
concentration of DMSO amounted to 0.1%). No effects of DMSO on the
anterior lobe neurons were observed.
The protein kinase inhibitors 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) and staurosporine and GF-109203X were obtained from Research Biochemicals International (Natick, MA). The phosphodiesterase-inhibitor IBMX was obtained from Sigma-Aldrich Chemie (Zwijndrecht, The Netherlands). H7, staurosporine, GF-109203X, and IBMX were dissolved in DMSO and diluted to a final concentration of 10, 1, 1, and 100 µM, respectively (final concentration of DMSO amounted to 0.1% in all cases). All agents were applied extracellularly by pressure ejection from a glass pipette.
Synthetic Lys-conopressin G (Cys-Phe-Ile-Arg-Asn-Cys-Pro-Lys-Gly-NH2) was obtained from Saxon Biochemicals (Hannover, Germany) and was applied by pressure ejection from a glass pipette. In all experiments, the recording chamber was continuously perfused with HBS.
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RESULTS |
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Stability of conopressin responses
Responses of individual anterior lobe neurons to conopressin
(applied at 1 µM) were characterized by testing the effect of the
peptide on their pseudo steady-state I-V relation, obtained with the use of voltage-ramp protocols. Most of the anterior lobe neurons (~60% of the cells) had
IHVA, which is activated by
conopressin at potentials positive to 40 mV. The isolated HVA current
manifested itself as a downward deflection of the steady-state
I-V relation at potentials positive to
40 mV (Fig.
1). Only some anterior lobe neurons (~10%) had
ILVA, which is activated by
conopressin at all potentials between
90 and +10 mV. The latter cells
always displayed combined activation of the LVA and HVA current by
conopressin, resulting in an inward shift of the entire I-V
relation between
90 and +10 mV (Fig. 3B).
As most of the experiments involved comparing responses to conopressin before and after pharmacological treatments, the ability to evoke reproducible responses was essential. Accordingly, the reproducibility of the conopressin-induced currents was investigated by repeatedly applying conopressin to the same cell. The applications were separated by approximately 20 min of washing, to allow the current to recover. Figure 1 shows the normalized amplitude of IHVA at each application (mean ± SD, number of cells indicated above each bar), indicating that no significant changes occur over a period of 1 h.
Involvement of cyclic AMP and PKA
Persistent inward currents in molluscan neurons have often been attributed to cAMP-dependent phosphorylation processes. To investigate whether the conopressin-induced persistent inward currents in Lymnaea anterior lobe neurons are also mediated by cAMP, several experimental approaches were followed.
Intracellular application of cAMP, by means of pressure ejection from
an intracellular microelectrode, induced an inward current similar to
that observed in other mollusks
(IcAMP; n = 5; Fig. 2A). Voltage ramps following long-lasting cAMP
injections revealed that the voltage dependence of
IcAMP showed large overlap with that
of the currents induced by conopressin (n = 5; Fig.
2B). However, because it is uncertain whether the
intracellular cAMP concentration was constant in time, the apparent
voltage dependence of IcAMP may be
distorted. Therefore similar experiments were performed using the
nonhydrolysable cAMP analogue dibutyryl-cAMP. First we measured the
time course of the current response on injection of dibutyryl-cAMP at a
constant holding potential of 60 mV. We found that the response
lasted for several minutes and that the current remained at a constant
amplitude for at least 60 s after injection (Fig. 2C;
n = 9). We then measured the pseudo steady-state I-V relation, using the voltage-ramp protocol between 30 and
50 s after injection. These I-V relations were very
similar to the ones obtained on injection of cAMP (Fig. 2D;
n = 5). In summary, the experiments demonstrate that
IcAMP is activated at a wide range of
potentials (from
90 to 0 mV). In this respect, it mostly resembles
the LVA current.
Since intracellular injection of cAMP could mimic at least part of the
conopressin response, we investigated whether the response required
cAMP. To this end, anterior lobe neurons were treated with the
phosphodiesterase-inhibitor IBMX. The resulting inhibition of cAMP
breakdown is expected to aid the presumed increase in cAMP levels, thus
potentiating the conopressin response if it were cAMP dependent. The
current induced by direct cAMP injection was potentiated by application
of 100 µM IBMX (Fig.
3A;
n = 5), indicating that the IBMX treatment was
effective. However, application of 100 µM IBMX had no effect on the
conopressin responses in five cells (Fig. 3B). The amplitude
of the HVA current (measured as the peak inward current, around 10
mV) amounted to 1.09 ± 0.26 nA under control conditions, and to
0.99 ± 0.19 nA after 15 min in the presence of IBMX (mean ± SD, P = 0.430). The LVA current was not affected
either; its amplitude (measured as the inward current at
50 mV) was
0.41 ± 0.08 nA under control conditions and 0.37 ± 0.14 nA
in the presence of IBMX (n = 4, P = 0.537). These data suggest that the conopressin-induced responses are not mediated by cAMP. The implication that also
ILVA (which resembles IcAMP) is not activated by cAMP is in
line with the observation that IcAMP
still could be evoked after complete desensitization of
ILVA (n = 5; data not
shown).
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As an additional control, we examined whether the response to
conopressin could still be evoked after desensitization of the response
to injection of cAMP. Desensitization of
IcAMP was induced by repeatedly
injecting a high concentration of cAMP (20 mM) into the cell until the
current amplitude approached zero (Fig.
4, middle). The
conopressin-induced currents, characterized using voltage ramps, were
recorded before and after desensitization of the cAMP response (Fig. 4,
left and right). Even after the response to
injection of cAMP had fully desensitized, conopressin could still
activate the HVA and LVA current. The amplitude of the HVA current
amounted to 0.92 ± 0.22 nA before and to 0.74 ± 0.38 nA
after desensitization of IcAMP
(n = 4; P = 0.436). For the LVA current
(measured at 50 mV), the values were 0.20 ± 0.20 nA before, and
0.26 ± 0.31 nA after the treatment (n = 4;
P = 0.439).
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These observations, together with the lack of effects of IBMX, suggest that cAMP elevation does not mediate conopressin-induced activation of neither the LVA current nor the HVA current. It may be, however, that cAMP and conopressin activate the same population of LVA channels in anterior lobe neurons, be it through different pathways.
Involvement of PKC
The sequence homology of the Lymnaea conopressin
receptors to mammalian vasopressin V1 and oxytocin receptors suggested
that the former might also couple to G proteins of the Q type
(Van Kesteren et al. 1995b, 1996
).
Therefore the involvement of PKC in the conopressin response was tested
by applying a phorbol ester that activates PKC in the absence of DAG.
In 11 cells exhibiting the HVA current on application of 1 µM
conopressin, extracellular application of the phorbol ester TPA (1 µM) induced a persistent inward current at voltages above 40 mV,
thus mimicking the activation of the HVA current by conopressin (Fig.
5). Furthermore, conopressin could not
induce additional inward current during the response to TPA
(n = 8). The occlusion of the conopressin-induced HVA
current by TPA suggests that both conopressin and TPA activate the same
population of ion channels.
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Application of 1 µM of the inactive phorbol ester 4--PMA, which
cannot activate PKC, did not induce current by itself, nor did it
prevent conopressin from activating the HVA current (n = 3, not shown). Subsequent application of TPA, however, affected the
cells as described above. These observations confirm that the observed
effect of TPA involves activation of PKC and is not due to nonspecific
effects on membrane components (see Hockberger et al.
1989
). In none of our experiments we observed a mimic of the
LVA current by TPA. Therefore the following pharmacological experiments
on the involvement of PKC in the conopressin response were restricted
to the HVA current.
PKC inhibitors
To demonstrate that activation of PKC is not only sufficient, but also necessary to activate the HVA current, we tested the effect of protein kinase inhibitors on the response induced by conopressin. The nonspecific protein kinase inhibitor H7 attenuated the conopressin response (not shown). After a 10-min application of 10 µM H7, the amplitude of the conopressin-induced HVA current was reduced by 25 ± 20% (from 0.76 ± 0.65 nA to 0.64 ± 0.72 nA; mean ± SD, n = 9, P = 0.012). A 20-min application of 100 µM H7 caused a similar reduction of the current amplitude by 32 ± 18% (from 0.73 ± 0.61 nA to 0.52 ± 0.56 nA; n = 5, P = 0.037). The current amplitude partially recovered after a 20-min wash out. This confirms that a protein kinase is involved in the conopressin response.
Subsequently, the effects of the membrane-permeable protein kinase inhibitor staurosporine were tested, both on the TPA-induced current and on the conopressin response. Staurosporine blocked the activation of the HVA current by 1 µM TPA (Fig. 6A). In five cells, the amplitude of the TPA-induced current was reduced by 76 ± 34% (from 1.43 ± 0.20 nA to 0.43 ± 0.33 nA; mean ± SD; Wilcoxon nonparametric test: P = 0.009). In addition, staurosporine largely blocked the HVA current response to conopressin. Figure 6B shows the response to 1 µM conopressin before and after application of 1 µM staurosporine. Comparable results were obtained with 1 and 10 µM staurosporine (n = 3 each); both treatments reduced the amplitude of the HVA current by 40-80% within 20 min. On average, the amplitude was reduced from 0.93 ± 0.82 nA to 0.30 ± 0.25 nA (n = 6, P = 0.028). However, because staurosporine (and H7) only have a limited specificity for PKC and also affect other kinases, we also tested the specific PKC inhibitor GF-109203X. At 1 µM, this inhibitor blocked the conopressin-evoked HVA current by 58 ± 7% after 15 min, and by 82 ± 4% after 35 min, reducing the HVA current amplitude from 0.87 ± 0.25 nA to 0.15 ± 0.39 nA in the latter case (Fig. 6C; n = 5, P = 0.015).
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These results, summarized in Fig. 6D, indicate that conopressin activates the HVA current through a PKC-dependent mechanism, probably phosphorylation of the ion channel or associated proteins.
Involvement of intracellular calcium
All experiments performed so far employed a pipette solution
containing 10 mM of the calcium chelator EGTA, buffered with calcium to
yield a final [Ca2+]in of
approximately 10 nM. The sole fact that the conopressin-induced currents are observed under these conditions (see also Van Soest and Kits 1997) argues against a strong involvement of changes in the intracellular free calcium concentration. To substantiate this
conclusion, we investigated the responses to conopressin using pipette
solutions containing either 0.1 mM EGTA, or 10 mM of the fast calcium
chelator BAPTA.
Due to its stabilizing effect on
[Ca2+]in, the presence of
10 mM EGTA might have obscured parts of the conopressin response that
were dependent on changes in
[Ca2+]in (e.g., due to
release of calcium from intracellular stores). Therefore the response
to conopressin was recorded with the use of a pipette solution
containing only 0.1 mM EGTA, but having the same concentration of free
calcium as the normal solution. With this pipette solution,
[Ca2+]in will be able to
vary substantially more. Under these circumstances, a strongly enhanced
and presumabably calcium-dependent outward current was observed at
depolarized voltages, interfering with the conopressin-induced
pacemaker currents. However, at voltages less than or equal to 20 mV,
conopressin still induced apparently normal LVA and HVA currents,
similar to the responses observed in standard saline (Fig.
7A;
n = 7). In another series of experiments, the low-EGTA
pipette solution was supplemented with cesium to block the outward
current and resolve better the conopressin- induced HVA current (Fig.
7B; n = 7). Again, apparently normal LVA and
HVA currents were observed.
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Subsequently, the response to conopressin was tested with a pipette solution containing 10 mM of the fast calcium chelator BAPTA, yielding a free calcium concentration of ~10 nM. Due to the fast calcium binding rate of BAPTA, transient variations in free calcium will be much less than with 10 mM EGTA. Figure 7C shows the responses to conopressin in a cell dialyzed with the BAPTA pipette solution. It is clear that conopressin is still capable of activating the HVA current, indicating that substantial changes in the intracellular calcium concentration are not necessary for the HVA current to occur. In the course of several tens of minutes, however, the amplitude of the current becomes progressively smaller (Fig. 7B), possibly suggesting that a calcium-dependent process is required for the long-term maintenance of (parts of) the signal transduction system.
Taken together, these experiments indicate that changes in the concentration of free calcium are not required for the conopressin-induced current responses. Apparently, synthesis of DAG suffices to activate PKC at this concentration of free calcium (around 10 nM; see also DISCUSSION), and thus to induce phosphorylation of the HVA channels or their associated proteins.
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DISCUSSION |
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Activation of PKC underlies HVA current
The present study demonstrates that activation of the HVA current
by conopressin is mediated by PKC. First of all, the HVA current
response is mimicked by application of the PKC-activating phorbol ester
TPA. During current activation by TPA, conopressin cannot activate
additional HVA current, indicating that both treatments affect the same
population of ion channels. These effects cannot be attributed to
nonspecific effects of TPA on, e.g., the ion channel itself (see
Hockberger et al. 1989), since the inactive phorbol
ester 4-
-PMA neither mimics nor occludes the HVA current response to
conopressin. Second, activation of the HVA current is inhibited by
staurosporine and H7, general protein kinase inhibitors with limited
preference for PKC, and by the specific PKC-inhibitor inhibitor
GF-109203X. As would be expected, the effect of TPA is also reversed by
application of staurosporine. On the basis of these results, we
conclude that activation of the HVA current is mediated by PKC. It
might be argued that this conclusion needs to be considered as
tentatively, because our inferences on the specificity of the various
pharmacological agents are based on biochemical data obtained in
vertebrates and Aplysia. Notably, between vertebrates and
invertebrates, this specificity may differ. For this reason we have
used several agents, either blocking, stimulating, or mimicking, the
effects of which all support the above stated conclusion.
The lack of effects of changing the calcium-buffering capacity in the
intracellular medium suggests that calcium dynamics do not play a
pivotal role in the signal transduction underlying the HVA current.
Apparently, PKC can be activated by DAG alone at the low level of
calcium (10 nM) imposed by 10 mM BAPTA. At a free calcium
concentration of 100 nM, DAG is known to suffice for activation of
mammalian PKC (Nishizuka 1984
).
Activation of LVA and HVA currents by conopressin involves different second messengers
The inward current that is activated in Lymnaea
anterior lobe neurons by direct injection of cAMP
(IcAMP) mostly resembles the
conopressin-induced LVA current: it is activated at potentials below
40 mV and its amplitude shows little dependence on the membrane
potential. In this respect, it bears resemblance to the cAMP-induced
current in Lymnaea buccal neurons (McCrohan and
Gillette 1988
).
However, the present results suggest that cAMP is not involved in activation of the LVA current by conopressin. The amplitude of the LVA current was unaffected by treatment with the phospodiesterase-inhibitor IBMX. In contrast, IcAMP was clearly augmented by IBMX. In itself, these observations may not be entirely conclusive, as it is possible that receptor activation would generate a saturating concentration of cAMP, and further increases due to reduced breakdown could not augment the response. However, the lack of cross-desensitization between the LVA current and the cAMP-induced current clearly confirms that they arise through different mechanisms. The present results are compatible with the idea that cAMP may induce activation of the same population of LVA channels as conopressin, be it through a parallel and independent pathway.
Our experiments argue against an effect of cAMP on the HVA current.
There is no cross desensitization between
IHVA and
IcAMP, there is no effect of IBMX on
IHVA, and cAMP does not mimic the HVA
response. Furthermore, because only activation of
IHVA involves PKC, whereas
ILVA is mimicked by cAMP, we assume
that conopressin activates ILVA and
IHVA through different signaling
pathways. This conclusion is in line with previous observations, that
the dose dependence and the rates of desensitization and wash out differ for the LVA and HVA current (Van Soest and Kits
1997).
Pacemaker currents in other mollusks
Our observation that activation of the HVA current is mediated by
PKC and not by PKA is not in line with previous studies on the actions
of vasopressin/oxytocin-related peptides in mollusks, that point to a
role of cAMP (e.g., Ifshin et al. 1975; Levitan et al. 1979
; Treistman and Levitan 1976
). For
example, the oxytocin-induced pacemaker current in an identified
Achatina neuron was concluded to be mediated by
cAMP-dependent phosphorylation, as the PKA inhibitors H8 and PKI
partially blocked the oxytocin response, while intracellular injection
of cAMP mimicked the response and IBMX augmented it (Funase
1990
). Interestingly, the oxytocin-induced current in the
Achatina neuron appears to be a composite of two currents, similar to the LVA and HVA currents in Lymnaea anterior lobe
neurons. Although the author does not make this distinction, his
results seem in line with the idea that PKA mainly underlies the LVA
part of the response.
Many other agonist-induced slow inward currents in molluscan neurons
have also been attributed to cAMP-dependent phosphorylation processes.
Still, the evidence regarding the transduction mechanisms underlying
many of these responses is inconclusive or even contradictory. For
instance, serotonin-induced currents have been attributed to direct
effects of cAMP (Price and Goldberg 1993),
phosphorylation by cAMP-dependent protein kinase (Funase et al.
1993
), an unknown cAMP-related mechanism (Kirk et al.
1988
), but also to a system completely independent of cAMP
(Kudo et al. 1991
).
The PKC-dependent HVA current in Lymnaea resembles to some
extent the nonselective cation current in bag cells, which is also activated by PKC (Wilson et al. 1998), but displays a
clear Ca dependence (Knox et al. 1996
; Wilson et
al. 1996
). Interestingly, this channel seems to cluster with
two different kinases (PKC and a tyrosine kinase) as well as a
phosphatase, indicating multiple phosphorylation mechanisms to act on
the channel (Wilson et al. 1998
).
Apparently, different signal transduction mechanisms may underlie similar responses in different cells or species. This may imply entirely different routes, involving different protein kinases such as PKA and PKC, toward the same type of channel, thus activating identical currents. Alternatively, if multiple types of ion channel with resemblant biophysical properties are present, different signal transduction routes may activate distinct ion channels giving rise to similar currents.
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ACKNOWLEDGMENTS |
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Present address of P. F. van Soest: Dept. of Developmental Neurobiology, Faculty of Biology, Vrije Universiteit, 1081 HV Amsterdam, The Netherlands.
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FOOTNOTES |
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Address for reprint requests: K. S. Kits, Dept. of Neurophysiology, Research Institute Neurosciences, Vrije Universiteit, De Boelelaan 1087, 1081 HV Amsterdam, The Netherlands (E-mail: ksk{at}bio.vu.nl).
Received 13 December 1999; accepted in final form 28 July 2000.
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REFERENCES |
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