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INTRODUCTION |
Angiotensin II (Ang II) has been recognized as an important neuropeptide in the CNS. Some of its functions are: regulation of baroreflex activity, modulation of blood pressure, regulation of water intake, and stimulation of arginine vasopressin secretion and norepinephrine release (Campagnole-Santos et al. 1988
; Phillips 1987
; Sumners et al. 1994
). These physiological actions of Ang II have been associated with the modulation of neuronal firing rates and firing patterns by working through the angiotensin type 1 (AT1) receptor subtype located on neurons in the hypothalamus and brain stem (Ambuhl et al. 1992
; Felix and Schlegel 1978
; Suga et al. 1979
; Yang et al. 1992
). However, the underlying mechanism(s) for this chronotropic action of Ang II has not been studied in isolated neurons.
Our previous studies showed that in neurons cultured from the rat hypothalamus and brain stem, activation of AT1 receptors by Ang II inhibits the delayed rectifier K+ current (IK) and stimulates calcium current (ICa). This action of Ang II was associated with phosphoinositide hydrolysis and protein kinase C (PKC) activation (Sumners et al. 1996
). Both the reduction in IK and the increase in ICa produced by Ang II were inhibited by PKC antagonists. In addition, the PKC activator phorbol 12-myristate 13-acetate (PMA) produced effects on IK and ICa similar to those of Ang II (Sumners et al. 1996
). Most recently we demonstrated that Ang II, via activation of AT1 receptors, reduced the transient potassium current (IA) and decreased the activity of a 15-pS, rapidly inactivating, 4-aminopyridine (4-AP)-sensitive K+ channel in these same neuronal cultures (see companion paper). Thus we propose that by potentiating ICa and diminishing IK and IA, Ang II increases the excitability of neurons from the hypothalamus and brain stem via an AT1 receptor.
In this study we have found that Ang II, by activation of AT1 receptors, increases neuronal excitability and firing frequency. These actions of Ang II include both PKC-dependent and -independent mechanisms.
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METHODS |
Materials
One-day-old Sprague-Dawley rats were obtained from our breeding colony, which originated from Charles River Farms (Wilmington, MA). Losartan potassium was generously provided by Dr. Ronald D. Smith of Du Pont-Merck (Wilmington, DE). PD 123319 was purchased from Research Biochemicals (Natick, MA). Dulbecco's modified Eagle's medium (DMEM) was obtained from GIBCO (Grand Island, NY). Crystallized trypsin (xl) was from Cooper Biomedical (Malvern, PA). Plasma-derived horse serum (PDHS), cytosine arabinoside (ARC), DNase I, poly-L-lysine (molecular weight 150,000), Ang II, ATP, guanosine 5
-triphosphate, PMA, tetraethylammonium chloride (TEA), 4-AP, tetrodotoxin (TTX), CdCl2, and N-2-hydroxyethylpiperazine-N
-2-ethanesulfonic acid (HEPES) were purchased from Sigma Chemical (St. Louis, MO). PKC inhibitory peptide (19-31, PKCIP) was purchased from Upstate Biotechnology (Lake Placid, NY). All other chemicals were purchased from Fisher Scientific (Pittsburgh, PA).
Preparation of neuronal cultures
Neuronal co-cultures were prepared from the brain stem and a hypothalamic block from 1-day-old Sprague-Dawley rats as described previously (Kang et al. 1992
). Trypsin (375 U/ml)- and DNase I (496 U/ml)-dissociated cells were resuspended in DMEM containing 10% PDHS and plated on poly-L-lysine-precoated 35-mm Nunc plastic tissue culture dishes. After cells were grown for 3 days at 37°C in a humidified incubator with 95% air-5% CO2, they were exposed to 1 µM ARC for 2 days in fresh DMEM containing 10% PDHS. Then ARC was removed and the cells were incubated with DMEM (10% PDHS) for a further 9-12 days before use. At the time of use, cultures consisted of 90% neurons and 10% astrocyte glia, as determined by immunofluorescent staining with antibodies against neurofilament proteins and glial fibrillary acidic proteins (Sumners et al. 1990
).
Electrophysiological recordings
Spontaneous and depolarizing pulse-elicited action potentials were recorded with the use of the whole cell voltage-clamp configuration in current-clamp mode (Hamill et al. 1981
). Experiments were performed at room temperature (22-23°C) with the use of an Axopatch-200A amplifier and Digidata 1200A interface (Axon Instruments, Burlingame, CA). Data acquisition and analysis were performed with the use of Axoscope 1.1 and pClamp 6.03. Cells were bathed in Tyrode's solution containing (in mM) 140 NaCl, 5.4 KCl, 2.0 CaCl2, 2.0 MgCl2, 0.3 NaH2PO4, 10 HEPES, and 10 dextrose, pH adjusted to 7.4 with NaOH. Neurons in the culture dish (volume 1.5 ml) were superfused at a rate of 2-4 ml/min. The patch electrodes (Kimax-5.1, Kimble Glass, Toledo, OH) had resistances of 3-4 M
when filled with an internal pipette solution containing (in mM) 140 KCl, 2 MgCl2, 4 ATP, 0.1 guanosine 5
-triphosphate, 10 dextrose, and 10 HEPES, pH adjusted to 7.2 with KOH. The whole cell configuration was formed by applying negative pressure to the patch electrode. A junction potential of
8 mV was corrected for all membrane potentials. The resting membrane potential was defined as the potential within a time period of 1 s during which there was no spontaneous action potential firing. The neuronal firing rate was measured as the numbers of fully developed action potentials per second (Hz). The subthreshold activity was defined as a depolarization from resting membrane potential without being fully developed into an action potential. The early afterdepolarization (EAD) was defined as a slow depolarization immediately following a fully developed action potential. In some instances, a large spike depolarization was superimposed on an EAD; these spikes are referred to as EAD-APs (Fig. 1). These EAD-APs vary widely in amplitude. Because an action potential must reach a certain amplitude to fulfill its signal conduction function, and in our neuronal system action potentials always had a peak depolarization beyond a membrane potential of 0 mV, we decided that if the EAD-AP amplitude went beyond a membrane potential of 0 mV, the EAD-AP would be treated as a fully developed action potential and would be counted in the firing rate. On the other hand, if a depolarization did not cross a membrane potential of 0 mV, it was treated as a nonfunctional event and was not included in the firing rate. The action potential amplitude was measured as the difference between the point of spike initiation and its peak amplitude. Because most of the cultured neurons had a prolonged EAD, we only reported the time from the action potential onset to the point at which the repolarization fell to half of the peak amplitude. In individual experiments, test agents were added sequentially in the superfusate. In the case of PKCIP, the agent was added to the patch pipette solution at the beginning of the experiment.

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| FIG. 1.
Effect of angiotensin II (Ang II) on firing rate of spontaneous action potentials in neuron (n = 7). A: control action potentials and subthreshold activities are indicated by arrows [early afterdepolarization (EAD), Foot Potential]. B: superfusion of Ang II (100 nM) increased firing rate and amplitude of EADs. Note that Ang II stimulated EAD to become fully developed action potential. C: action of Ang II was reversed by selective angiotensin type 1 (AT1) receptor antagonist losartan (1 µM). Losartan alone had no effect when tested in other cells (n = 3). Calibration bars in C apply to all records. Horizontal dashed line: membrane potential of 0 mV.
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Data analysis
Results are expressed as means ± SE. Statistical significance was evaluated with the use of paired Student's t-test. Differences were considered significant at P < 0.05; n corresponds to the number of cells examined.
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RESULTS |
Spontaneous action potentials were observed in neurons grown in culture from rat hypothalamus and brain stem (Fig. 1). The action potential parameters were similar to those described previously by others (Williams 1996; Zhang and McBain 1995
). At room temperature (22-23°C), the mean amplitude and point at which repolarization fell to half of peak amplitude for these action potentials were 86 ± 2.7 (SE) mV and 2.3 ± 0.2 ms, respectively (n = 26). The apparent resting membrane potential of these neurons had a mean value of
59.2 ± 3.1 mV. Only a few cells showed burst (or rhythmic) firing patterns, which were not subject to further experimental observation. The spontaneous action potentials fired at a mean frequency of 0.8 ± 0.1 Hz (n = 32, range from 0.3 to 3 Hz). Most of the cells had a prolonged EAD that followed an initial fully developed action potential. The spontaneous action potentials could be completely abolished by TTX (100 nM). All the experiments were carried out in the presence of PD 123319 (1 µM) to block AT2-receptor-mediated responses. PD 123319 (1 µM) alone had no effect on the parameters measured.
Action of Ang II on spontaneous action potentials
Figure 1 demonstrates the different types of electrical activity measured in this study. Figure 1 presents the records from a single neuron that is representative of the positive chronotropic effect of Ang II. These records include action potentials; EADs (Fig. 1, A and B); spontaneous action potentials triggered by EADs (Fig. 1B, indicated by EAD-AP); and subthreshhold oscillations that produce foot potentials that trigger action potentials (Fig. 1A). Superfusion of Ang II (100 nM) significantly increased the neuronal firing rate (Fig. 1). This concentration of Ang II produces a maximum effect when tested on individual ionic currents. This stimulatory action of Ang II on neuronal electrical activity was completely reversed by addition of the AT1 receptor antagonist losartan (1 µM, Fig. 1). Losartan alone had no effect on neuronal electrical activity (data not shown). On average, the spontaneous firing rate was increased from0.8 ± 0.3 in control to 1.3 ± 0.4 Hz in the presence of Ang II (n = 7, P < 0.05). This 62% increase in firing rate should result in a large absolute increase in firing rate in vivo at 38°C. To determine whether Ang II increases the excitability of the cultured neurons, the threshold current amplitude needed to elicit an action potential was studied in a group of neurons that did not fire action potentials spontaneously. Although Ang II did not significantly affect the resting membrane potential, the amplitude of the stimulus current needed to reach threshold was decreased by Ang II (100 nM) from 82 ± 4 pA to 62 ± 5 pA (n = 4, P < 0.05). Superfusion of losartan (1 µM) reversed the effect of Ang II (Fig. 2). These data demonstrate that Ang II increases the excitability of neurons in culture via the AT1 receptor.

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| FIG. 2.
Effect of Ang II on level of threshold current required to elicit fully developed action potential. Representative neuron (n = 4) was stimulated by current pulses (2 ms in duration) at rate of 1 Hz. Pulse amplitude was increased from 50 to 80 pA in steps of 10 pA to determine threshold current amplitude needed to activate cell. Numbers near traces: responses elicited by current pulses with corresponding amplitude (1 = 50, 2 = 60, 3 = 70, 4 = 80 pA, respectively). A: in control, threshold current amplitude was 80 pA. B: Ang II (100 nM) decreased amplitude of threshold current to 60 pA. C: effect of Ang II on threshold current was reversed by losartan (1 µM). Calibration bars at bottom apply to all records. Horizontal dashed line: membrane potential of 0 mV.
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Ang II increased the subthreshold activities in the cultured neurons
The two types of subthreshold activities shown in Fig. 1 were EADs and subthreshold oscillations. The latter type occurred at the resting membrane potential and oscillated the membrane potential in a depolarizing direction. When the depolarization amplitude of the oscillation was below the threshold potential, the oscillation was manifested as a subthreshold spike as shown in Figs. 3A, 4, A and C, and 6C (indicated by arrows). If the oscillation reached the threshold potential, it composed the foot potential of a fully developed action potential as shown in Fig. 1A. The second type of subthreshold activity occurred before an action potential completely repolarized. This activity was similar to the EAD described in cardiac myocytes (Zeng and Rudy 1995
).

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| FIG. 3.
Effect of Ang II on subthreshold oscillations in representative neuron (n = 5). A: control spontaneous action potentials. B: tetrodotoxin (TTX, 100 nM) abolished spontaneous action potentials. C: addition of Ang II (100 nM) increased subthreshold oscillations (as indicated by arrows) in presence of TTX. D: effect of Ang II on subthreshold activities was reversed by Cd2+ (10 µM). Calibration bars in D apply to all records. Horizontal dashed line: membrane potential of 0 mV.
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| FIG. 4.
Effects of Cd2+ on firing rate of spontaneous action potentials in representative neuron (n = 4). A: control action potentials, subthreshold activities, and EAD triggered action potentials are indicated by arrows. B: superfusion of Cd2+ (10 µM) reduced firing rate and eliminated subthreshold activities. C: effect of Cd2+ was reversed on washout. D: superimposed action potentials (recorded at fast speed) show that foot potential and EAD (indicated by arrows on  ) were eliminated by Cd2+ (· · ·). Calibration bars in C also apply to records in A and B. Horizontal dashed line: membrane potential of 0 mV.
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| FIG. 6.
Effect of PMA on subthreshold oscillations in representative neuron (n = 5). A: control spontaneous action potentials. B: TTX (100 nM) abolished spontaneous action potentials. C: addition of PMA (100 nM) increased subthreshold oscillations in presence of TTX. D: effect of PMA on subthreshold activities was reversed by Cd2+ (10 µM). Calibration bars in D apply to all records. Horizontal dashed line: membrane potential of 0 mV.
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The effect of Ang II on EAD is shown in Fig. 1. Superfusion of Ang II (100 nM) increased the amplitude and duration of EADs (compare Fig. 1, A and B). Ang II also stimulated the subthreshold EADs to become fully developed action potentials (Fig. 1, indicated by EAD-AP). To determine whether Ang II increases the subthreshold oscillations, we used TTX (100 nM) to inhibit the spontaneous action potentials (Fig. 3B). In the presence of TTX, Ang II (100 nM) significantly increased the frequency and the amplitude of the subthreshold oscillations (Fig. 3C). This action of Ang II was inhibited by Cd2+ (Fig. 3D). Our previous studies demonstrated that superfusion of Ang II (100 nM) results in a significant stimulatory effect on neuronal ICa, which is mediated by AT1 receptors (Sumners et al. 1996
). To determine whether these subthreshold activities are related to Ca2+ channels, we examined the action of Cd2+ (a Ca2+ channel blocker) on the spontaneous action potential firing rate. As shown in Fig. 4, superfusion of Cd2+ (10 µM) markedly reduced the firing rate. The decrease in the firing rate was associated with the elimination of the EADs and the foot potentials (Fig. 4, B and D). The effects of Cd2+ were reversible on washout. These data indicate that the subthreshold activities (both EADs and baseline oscillations) are closely related to the generation of spontaneous action potentials in cultured neurons and were mediated by Ca2+ influx through Cd2+-sensitive Ca2+ channels. One possible interpretation of the data shown in Fig. 1 is that Ang II stimulates the firing rate of action potentials by enhancing these subthreshold neuronal activities.
PKC increases neuronal firing
Recently, Sumners et al. (1996)
demonstrated that the PKC activator PMA produced an effect on ICa and IK similar to that elicited by Ang II. Figure 5 shows a representative experiment with PMA. Superfusion of PMA (100 nM) increased the firing rate from 0.76 ± 0.3 Hz to 2.3 ± 0.2 Hz (n = 6, P < 0.05). Interestingly, addition of Ang II (100 nM) with PMA resulted in a further increase in the firing rate to 3.9 ± 0.4 Hz (n = 4, P < 0.05, Fig. 5C).

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| FIG. 5.
Effect of phorbol 12-myristate 13-acetate (PMA) on firing rate of neuronal spontaneous action potentials in representative neuron (n = 5). Numbers at right: firing rate (Hz) for this neuron. A: control recording was made before application of PMA. B: superfusion of PMA (100 nM) markedly increased firing rate from 0.73 to 2.13 Hz. C: addition of Ang II (n = 4, 100 nM) further increased firing rate in presence of PMA. Calibration bars in C apply to all records. Horizontal dashed line: membrane potential of 0 mV.
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In Fig. 6, we present a typical example of the effect of PMA on subthreshold oscillations. Similar to the action of Ang II, PMA could increase the subthreshold activity after the spontaneous action potentials were eliminated by TTX (100 nM, Fig. 6, B and C). To further elucidate that the PKC activation was involved in the intracellular mechanism(s) of the Ang II-stimulated increase in neuronal activity, we tested the actions of Ang II and PMA on neuronal firing rate when the cells were intracellularly dialyzed with PKCIP. A representative cell is shown in Fig. 7. PMA alone (Fig. 7B), when tested in seven cells, increased the firing rate from 0.76 to 2.3 Hz. In the presence of PKCIP, the PMA-triggered increase in the firing rate from 0.52 ± 0.24 Hz to 0.67 ± 2.3 Hz was not significant (n = 4, P > 0.05). In the same four cells, Ang II was added to PMA in the presence of PKCIP (Fig. 7C) and it significantly increased the firing rate to 0.98 ± 0.31 Hz (P < 0.05). A similar result was obtained in cells exposed only to Ang II in the presence of PKCIP in the patch pipette (data not shown). Consistent with these results, PKCIP also abolished the stimulatory actions of Ang II and PMA on subthreshold oscillations in the presence of TTX (data not shown). This result was consistent with the observations made by Sumners et al. (1996)
that PKCIP abolished the actions of PMA on the stimulation of ICa and the decrease in IK. However, superfusion of PMA plus Ang II (Fig. 7C, inset) and Ang II alone (in other cells not shown) increased the number of EADs that reached threshold to fire action potentials in the presence of PKCIP. These results plus those presented in Fig. 5C are consistent with the possibility that Ang II may have the potential to work through a PKC-independent pathway when PKC is inhibited.

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| FIG. 7.
Effect of PMA and Ang II on firing rate of neuronal spontaneous action potentials in presence of protein kinase C inhibitory peptide (PKCIP, predissolved in pipette solution). These records are from representative neuron(n = 4). Recordings at right: expansion of circled regions. A: control action potentials, EAD, and EAD action potentials (EAD-APs) recorded after cell was dialyzed with PKCIP (5 µM). B: PMA (100 nM) did not increase firing rate in presence of PKCIP. C: Ang II (100 nM) stimulated EADs to become fully developed action potentials in presence of PKCIP. Calibration bars in C, left, apply to all records at left; calibration bars in C, right, apply to all records at right. Horizontal dashed line: membrane potential of 0 mV.
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Actions of depolarizing pulse, hyperpolarizing pulse, TEA, and 4-AP on spontaneous action potentials of cultured neurons
The firing rate of spontaneous action potentials in cultured neurons could be markedly accelerated by application of a +30-pA depolarizing pulse. The more the cell was depolarized, the higher the firing frequency became. The neurons showed a frequency adaptation when the depolarizing potential was >80 pA (data not shown). In contrast, application of a hyperpolarizing current pulse (
30 pA) caused the cell to become quiescent.
It is well known that potassium channels play a major role in determining the characteristics of neuronal repolarization and resting potential (Hille 1992
). Thus the following studies were designed to elucidate the role of these channels in the firing patterns of cultured neurons. Superfusion of the potassium channel blockers TEA or 4-AP altered the neuronal firing rate, but in two different manners. TEA (3 mM) increased the firing rate from 0.54 ± 0.27 Hz to 1.2 ± 0.26 Hz (Fig. 8B) and lengthened the time for 50% repolarization from 2.2 ± 0.1 ms to 2.4 ± 0.2 ms (Fig. 8C). The prolongation of the action potential induced by TEA mainly occurred at the more repolarized (negative) potentials (Fig. 8C), whereas the initial rate of repolarization was not noticeably affected (Fig. 8C, · · ·). High concentrations of TEA (>10 mM) or prolonged exposure of the cells to TEA (>2 min) resulted in a frequency-adaptation-like suppression of the spontaneous firing of neuronal action potentials (data not shown).

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| FIG. 8.
Effect of tetraethylammonium chloride (TEA) on firing rate and repolarization of action potentials in representative neuron (n = 3). A: control action potentials, EADs, and EAD action potentials. B: superfusion of TEA (3 mM) increased neuronal firing rate. C: superimposed recordings made at fast speed show that TEA induced prolongation in action potential duration mainly occurred after cell reached 50% of repolarization. Calibration bars in B apply to record in A. Horizontal dashed line: membrane potential of 0 mV.
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4-AP (2.5 mM) affected the spontaneous neuronal activity by increasing the firing rate from 0.55 ± 0.23 Hz to 1.1 ± 0.22 Hz (Fig. 9B) and subsequently prolonged the action potential duration from 2.1 ± 0.1 ms to 3.5 ± 0.2 ms (Fig. 9C). In contrast to TEA, the prolongation of action potential duration induced by 4-AP started immediately during the repolarization phase (Fig. 9C, · · ·).

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| FIG. 9.
Effect of 4-aminopyridine (4-AP) on firing rate and repolarization of action potentials in representative neuron (n = 4). A: control action potentials, EADs, and EAD action potentials. B: superfusion of 4-AP (2.5 mM) increased neuronal firing rate. C: superimposed recordings made at fast speed show that 4-AP-induced prolongation of action potential occurred throughout repolarization. Calibration bars in B apply to record in A.
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DISCUSSION |
In this study we demonstrate that activation of AT1 receptors results in an increase in the firing rate of spontaneous action potentials in neurons cultured from newborn rat hypothalamus and brain stem. Consistent with this chronotropic effect, Ang II also increased the subthreshold oscillations and decreased the threshold current amplitude needed to elicit an action potential in these cells. We also found that the chronotropic actions of Ang II, at least in part, involve a PKC-mediated mechanism and are related to the increase in the influx of Ca2+.
The data presented here integrate well with our previous studies on the regulation of transmembrane currents in these cells by Ang II (Kang et al. 1992
; Sumners et al. 1996
; see companion paper). By attenuation of IK and IA and enhancement of ICa, Ang II via activation of AT1 receptors decreases the net outward current, namely Ino. The overall effect of the decrease in Ino (decreased IA and IK, increased ICa) is to increase neuronal excitability. This is manifested as an increase in the spontaneous firing rate as well as in the occurrence of larger subthreshold oscillations. Further, AT1 receptor stimulation results in an increase in the amplitude of EADs and EAD-triggered action potentials.
As we reported in the companion paper, there are at least four kinds of ionic currents that can be recorded from the cultured neurons we used in this study. Two of them are inward Na+ (TTX-sensitive) and Ca2+ (Cd2+-sensitive) currents. The other two are outward K+ currents, IK (TEA sensitive) and IA (4-AP sensitive). Alteration of any one of these currents could affect the neuronal excitability such that there would be an increase in firing rate, subthreshold oscillations, and EADs in the cultured cells. The increase in the inward current (ICa) caused by a depolarizing pulse (Fig. 8) or by Ang II or PMA stimulates the neurons to fire action potentials, whereas blockade of the inward currents (INa by TTX and ICa by Cd2+) decreases neuronal spontaneous discharges (Figs. 3 and 4).
We believe that the action potentials fired from the resting potential level are due to spontaneous activation of INa. Evidence that supports this argument includes the following: 1) spontaneous action potentials can be completely eliminated by TTX and 2) the study of the activation and inactivation of INa in these neurons revealed a significant window current of INa within a membrane potential range between
60 and
50 mV (unpublished observations). It has been reported that activation of PKC can modulate INa (Cantrell et al. 1996
; Godoy and Cukierman 1994
; Numann et al. 1991
). Numann et al. (1991)
and Cantrell et al. (1996)
demonstrated an inhibitory effect of PKC, whereas Godoy and Cukierman (1994)
found both inhibitory and stimulatory effects mediated by PKC in mouse neuroblastoma cells. However, whether the action of Ang II on neuronal firing rate involves a direct modulation of the Na+ channel needs to be studied further.
The role of ICa in the firing of action potentials has been well documented (Meyers 1993
; Penington et al. 1992
). Here, we clearly illustrated that the Cd2+-sensitive ICa is closely associated with the subthreshold oscillation and EADs. It is possible that the two types of subthreshold activities are mediated by different subtypes of ICa. The subthreshold oscillation recorded at the resting membrane potential level might be related to activation of the low-threshold (T-type) Ca2+ current (ICa,T). Indeed, ICa,T has been observed in these cultured neurons, when activated by depolarizing pulses from
80 to
50 mV (unpublished observations). However, additional experiments in which specific blockers and pulse protocols are used are planned to delineate the mechanism of interaction of Ang II and these subthreshold oscillations. The EADs following most action potentials may be due to activation of high-threshold (L-type) Ca2+ current (ICa,L). However, as with the subthreshold oscillations, further studies are planned to test this hypothesis. At the present time, this hypothesis is based on the long time course of the EADs, which is consistent with the involvement of ICa,L. These afterdepolarizations may be functionally similar to those seen in the heart (Wit and Rosen 1986
) in that they can summate and lead to a triggered increase in automatic firing rate. Thus, under the influence of Ang II, they could underlie the increases seen in neuronal firing patterns in vivo.
Ang II caused a 15% decrease in IK and a 20% decrease in IA in the cultured neurons (see companion paper); the combined decrease in these outward K+ currents probably plays an important role in the stimulatory action of this peptide on cell excitability. The increase in the neuronal firing rate caused by TEA and 4-AP that mimics the action of Ang II provides the evidence that decreasing outward K+ currents alone can increase neuronal excitability. We have observed that blockade of IK by TEA or IA by 4-AP indeed increased the magnitude of net inward current. Thus, by stimulation of inward ICa and at the same time inhibiting IK and IA, Ang II produces its stimulatory chronotropic action on neuronal activity.
In our cultured neurons, activation of AT1 receptors results in a stimulation of phosphoinositide hydrolysis (Sumners et al. 1994
) that results in production of diacylglycerol (which activates PKC) and inositol-(1,4,5)-triphosphate (IP3; which triggers the release of Ca2+ from intracellular stores) (Berridge 1988
). Activation of PKC can modulate several channel activities (Levitan 1994
; Stea et al. 1995
; Sumners et al. 1996
). For instance, the modulation of both IK and ICa by Ang II was completely abolished by the PKC antagonist (PKCIP, 3 µM), indicating an involvement of PKC activation in these responses (Sumners et al. 1996
). Consistent with the modulation of Ang II on IK and ICa, the positive chronotropic effect of Ang II also involved the activation of PKC. This statement is supported by the data that demonstrate that the PKC activator PMA also stimulated the firing rate and subthreshold oscillation in cultured neurons (Figs. 5 and 6). PKCIP, at a concentration of 5 µM, which abolished the effects of PMA on IK, ICa, and firing rate (Sumners et al. 1996
), only inhibited the Ang II-mediated stimulation of the firing of action potentials from the resting membrane potential level but did not affect the Ang II-stimulated firing of action potentials from EADs (Fig. 7C, inset). On this basis we propose that the stimulatory action of Ang II on EAD-triggered neuronal firing may involve other intracellular mechanism(s) that are PKC independent. In our cell-attached patch-clamp experiments (see companion paper) we demonstrate that Ang II can decrease the A-type K+ channel activity through an intracellular signal transduction pathway. The inhibitory action of Ang II on IA may also be PKC independent.
In summary, activation of AT1 receptors by Ang II results in an increase in the firing rate of spontaneous action potentials in cultured neurons. Consistent with this effect, Ang II also decreases the threshold current needed to elicit an action potential and increases the subthreshold oscillations that are dependent on the increase in ICa. We also demonstrated that these actions of Ang II involved both a PKC-mediated and a PKC-independent mechanism in these cells.