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Volume 17, Number 22, Issue of November 15, 1997

Nociceptin Inhibits T-Type Ca²⁺ Channel Current in Rat Sensory Neurons by a G-Protein-Independent Mechanism

Fuad A. Abdulla and Peter A. Smith

Department of Pharmacology, University of Alberta, Edmonton, Alberta, Canada, T6G 2H7

ABSTRACT

INTRODUCTION

MATERIALS AND METHODS

RESULTS

DISCUSSION

FOOTNOTES

REFERENCES

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ABSTRACT

Nociceptin (orphanin FQ) is a novel, opioid-like, heptadecapeptide that is an endogenous ligand for the opioid receptor-like (ORL₁) receptor. Unlike classical opioids, nociceptin can produce hyperalgesia when injected intracerebroventricularly into mice. Despite this, nociceptin has been reported to decrease transmitter release, activate an inwardly rectifying K⁺ conductance, and suppress high-voltage-activated Ca²⁺ channel conductances (HVA g_Ca) in much the same way as µ-, -, and -opioids. We report an action of nociceptin that is not shared by morphine: the suppression of low-voltage-activated, transient calcium (barium) current (I_Ba,T) in acutely dissociated rat dorsal root ganglion (DRG) neurons (EC₅₀ = 100 nM). This effect was reflected as inhibition of bursts of action potentials that can be evoked in "medium-sized" DRG neurons. Experiments with GTP--S (100 µM), GDP--S (2 mM), or aluminum fluoride (AlF₃) (100 µM) in the patch pipette failed to provide evidence for G-protein involvement in nociceptin-induced I_Ba,T suppression. By contrast, both morphine and nociceptin suppressed HVA g_Ca, and the latter response was affected by intracellular GTP--S, GDP--S, and AlF₃ in ways that confirmed G-protein involvement. The selective effect of nociceptin on I_Ba,T may therefore be relevant to understanding why its behavioral actions differ from those of other opioids. This G-protein-independent effect of the action of nociceptin may reflect a new general mechanism of action for opioid peptides within the nervous system.

Key words: LC132 receptor; neuropathic pain; sympathetic ganglion; orphan receptor; enkephalin; endorphin; spinal cord; afterdepolarization; anticonvulsant

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INTRODUCTION

The heptadecapeptide nociceptin (orphanin FQ) is an endogenous ligand for the opioid receptor-like (ORL₁) receptor (Meunier et al., 1995; Reinscheid et al., 1995; Nothacker et al., 1996). This substance has attracted considerable attention, because unlike conventional opioids it does not produce analgesia when injected intracerebroventricularly into mice; in fact, nociceptin produces hyperalgesia in a tail-flick assay (Meunier et al., 1995; Reinscheid et al., 1995). Despite this, the cellular actions of nociceptin appear similar to those of µ-, -, and -opioids. Opioids suppress neurotransmitter release (Jessell and Iversen, 1977; MacDonald and Nelson, 1978; Cherubini and North, 1985; Hori et al., 1992; Vaughan and Christie, 1997), and µ-, -, or -opioids attenuate high-voltage-activated Ca²⁺ channel conductances (HVA g_Ca) (Hescheler et al., 1987; Surprenant et al., 1990; Schroeder et al., 1991; Moises et al., 1994a,b; Womack and McCleskey, 1995) and activate an inwardly rectifying K⁺ conductance (g_KIR) (North et al., 1987; Williams et al., 1988; Grudt and Williams, 1993). Similarly, nociceptin suppresses transmitter release in the periaqueductal gray matter (Vaughan et al., 1997) and in the superficial dorsal horn of neonatal rat spinal cord (Liebel et al., 1997). It also inhibits HVA g_Ca in hippocampal neurons (Knoflach et al., 1996) and in a neuroblastoma cell line (Connor et al., 1996b). Nociceptin activates g_KIR in dorsal raphe neurons (Vaughan and Christie, 1996), in midbrain periaqueductal gray neurons (Vaughan et al., 1997), and in locus coeruleus neurons (Connor et al., 1996a).

The molecular mechanisms underlying opioid actions are often studied by examining their effects on g_Ca in the cell bodies of sensory neurons that lie in dorsal root ganglia (DRG) (Schroeder et al., 1991; Moises et al., 1994a,b; Wilding et al., 1995; Womack and McCleskey, 1995). We report here an action of nociceptin on rat DRG neurons that is not shared by the classic analgesic opioid morphine. Although both agonists suppress HVA g_Ca by a G-protein-dependent mechanism, only nociceptin affects the low-voltage-activated (LVA), transient I_Ca (T-current, I_Ca,T). This effect, which does not seem to involve G-protein activation, is reflected in the attenuation of action potential afterdepolarization and multiple spike discharge in "medium-sized" DRG cells. Whereas I_Ca,N is normally associated with neurotransmitter release (DeWaard et al., 1996), I_Ca,T is responsible for generating repetitive or bursting discharges, for boosting Ca²⁺ entry, and for generation of low-threshold spikes (Huguenard, 1996). The selective suppression of I_Ca,T by nociceptin may therefore be relevant to understanding why its behavioral actions differ from those of morphine. This novel action supports the idea that certain effects of opioid peptides may be generated without the involvement of G-proteins (Twitchell and Rane, 1994; Brauneis et al., 1996).

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MATERIALS AND METHODS

Male Sprague Dawley rats (180-300 gm) were decapitated in a small animal guillotine, and neurons from L₄ and L₅ DRG were dissociated using a trypsin/collagenase/DNase/trypsin inhibitor procedure as described by White et al. (1989). Dissociated cells were plated into petri dishes, superfused with various extracellular solutions at ~2 ml/min, and used for recording within 2-10 hr. Whole-cell voltage recordings were made using an Axoclamp 1B amplifier in bridge-balance mode, and action potentials (APs) were evoked by passing 2-5 msec depolarizing currents through the recording electrode. Single-electrode, discontinuous voltage-clamp mode was used to record I_Ca with Ba²⁺ as charge carrier (I_Ba). Borosilicate glass patch electrodes had DC resistances of 4-6 M for AP recording or 1-3 M for current recording. Because the dissociated cells were relatively small and spherical, currents of 10 nA or more could be recorded with little or no voltage control problems. I_Ba tails recorded at 40mV were described by a single exponential function. Because Ba²⁺ was used as a charge carrier, the slow tail currents that result from activation of Ca²⁺-dependent Cl conductances (g_Cl,Ca) (Mayer, 1985) were not observed. A P/4 leak subtraction paradigm was used when necessary.

Under bridge-balance current-clamp, input capacitance (C_in) was calculated from the input resistance (R_in) and the membrane time constant (_m) using the equation _m = C_inR_in. Under voltage clamp, C_in was measured by integrating the area of capacitative current transients that were generated by 10 mV commands (V). This yielded the charge Q that is related to C_in by Q = VC_in. DRG neurons were classified into three groups according to their size and AP shape. "Large" cells were defined as those with AP duration <3 msec and C_in >90 pF; "medium" cells had an AP duration of 3-5 msec and C_in of 70-90 pF; and "small" cells had an AP duration >5 msec and C_in <70 pF. Both medium and small cells exhibited a "hump" or "shoulder" on the falling phase of their AP (Abdulla and Smith, 1997). Spike duration (AP width) was measured at half-maximum amplitude. Because T-currents (I_Ba,T) were encountered most frequently in the medium-sized cells (Scroggs and Fox, 1992), all experiments were performed on cells within the 70-90 pF size range, 40% of which had a robust I_Ba,T. Only cells with "giant" I_Ba,T (>1 nA) (Rusin and Moises, 1995) were selected for analysis.

For AP recording, external solution contained (in mM): 150 NaCl, 5 KCl, 2.5 CaCl₂, 1 MgCl₂, 10 HEPES-NaOH, pH 7.4, and 10 D-glucose; osmolarity, 330-340 mOsm. Internal solution contained (in mM): 130 potassium gluconate, 2 Mg-ATP, 0.3 Na-GTP, 11 EGTA, 10 HEPES-KOH, pH 7.2, and 1 CaCl₂; osmolarity, 310-320 mOsm. For recording LVA and HVA I_Ba, external solution contained (in mM): 160 TEA-Cl, 10 HEPES, 2 BaCl₂, 10 glucose, and 200 nM TTX, adjusted to pH 7.4 with TEA-OH; internal solution contained (in mM): 120 CsCl₂, 5 Mg-ATP, 0.4 Na-GTP, 10 EGTA, 20 HEPES-CsOH, pH 7.2. Except where stated otherwise, I_Ba,T was recorded in response to a command to 40 mV, and HVA currents (N, L, P, and presumptive Q types) (Rusin and Moises, 1995, DeWaard et al., 1996) were activated by commands to 10 mV. The holding potential (V_h) for voltage-clamp experiments was 90 mV.

Drugs were applied by bath superfusion using a tap system that had an exchange time of ~90 sec. Nociceptin (rat or human; Phe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys-Leu-Ala-Asn-Gln) was from Peptide Institute Inc. (Louisville, KY); naloxone hydrochloride, naloxone benzoylhydrazone (nalbzoh), and guanosine-5-O-(3-thiodiphosphate) (GTP--S; trilithium salt) were from Research Biochemical International (Natick, MA), and guanosine-5-O-(2-thiodiphosphate) (GDP--S; trilithium salt) was from Calbiochem (San Diego, CA) or from RBI. All other chemicals were from Sigma (St. Louis. MO).

Data were acquired and analyzed using Pclamp software (version 5.5.1), and final figures, except for Figure 6A, were produced using Origin 4.10 (Microcal).

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Fig. 6. Biophysical aspects of nociceptin-induced T-current suppression. A, Slow speed chart record to show rate of onset and the effect of nociceptin with and without repeated activation of g_Ba,T. Top record is current, and bottom record is voltage. Deflections result from brief (30 msec) voltage commands to 40 mV from the holding potential of 90 mV applied once every 2.5 sec (0.4 Hz). Nociceptin (1 µM) was first applied (at left) while g_Ba,T was repeatedly activated. The current deflections attain a smaller steady-state amplitude 3 min after initiating nociceptin superfusion. Nociceptin was reapplied (at right) in the absence of g_Ba,T activation. Voltage commands were recommenced 3 min after initiating nociceptin superfusion, and I_Ba,T responses were attenuated to the same extent as those recorded when the conductance had been activated repetitively in the presence of nociceptin. B, Normalized steady-state inactivation plots for g_Ba,T acquired before and during application of 100 nM nociceptin. Cell was held at a series of prepulse potentials (+5 mV increments from 115 mV) for 200 msec before activating g_Ba,T at 40 mV. Voltage protocol is shown as inset. C₁, C₂, Original data records used to produce the data in B. A split-clock protocol was used so that 100 msec calibration refers to prepulse and 25 msec calibration refers to recordings of I_Ba,T.
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RESULTS

Suppression of T-current by nociceptin

Nociceptin (0.01-1.0 µM) suppressed I_Ba,T in all cells tested (n > 100). Figure 1A₁ illustrates almost complete suppression of I_Ba,T by 1 µM nociceptin in a medium-sized DRG neuron. An equal concentration of morphine produced only a small (<10%) suppression of I_Ba,T in the same cell (Fig. 1A₂). Morphine (up to 10 µM) produced no more than 20% suppression of I_Ba,T in 18 of 67 cells tested, and the remainder (49) were unaffected. Nociceptin-induced inhibition was reflected in attenuation of I_Ba,T tails recorded at 60 mV (Fig. 1B).

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Fig. 1. Blockade of I_Ba,T by nociceptin. A₁, A₂, Comparison of the effects of nociceptin and morphine on I_Ba,T in the same neuron. Voltage record (step to 40 mV from a holding potential of 90 mV) omitted for clarity. Large capacity current transients accompanying voltage steps have been attenuated or removed by erasing three or more data points (clock speed for data acquisition was 0.05 msec/point). A₁, Superimposed records to show almost complete suppression of I_Ba,T by 1 µM nociceptin. A₂, In the same cell, 1 µM morphine suppresses the current by <10%. B, Superimposed records obtained from another cell to show suppression of I_Ba,T tails by 1 µM nociceptin. Tails recorded at 60 mV after brief 30 msec step to 40mV from a holding potential of 90 mV (voltage record omitted for clarity). C, I-V relationships for I_Ba recorded in the absence and presence of 1 µM nociceptin. Asterisk marks characteristic shoulder on I-V relationship that results from I_Ba,T. Note attenuation of current at 40 mV (I_Ba,T) and 10 mV (attenuation of HVA I_Ba) by nociceptin. D, I-V relationships for I_Ba recorded in the absence and presence of 1 µM morphine. Note lack of attenuation of current at potentials more negative than 40 mV (I_Ba,T, marked by asterisk) and attenuation of HVA I_Ba recorded at potentials positive to 20 mV. E, Log-concentration-response relationship for nociceptin. Numbers on graph indicate numbers of cells tested with each concentration; error bars, which indicate SEM, are smaller than data markers in some cases. The concentration of nociceptin required for half-maximum suppression of I_Ba,T (EC₅₀) is 100 nM. F, Data from E replotted as a Hill plot [Log₁₀ R/R  R_m vs Log₁₀ nociceptin concentration  EC₅₀; where R is amplitude of response and R_m is the maximum response that can be obtained; K_D, the dissociation equilibrium constant for nociceptin and its receptor is assumed to equal the EC₅₀ (100 nM)]. Linear fit to data points yields a line with a gradient of 1 (Hill coefficient of 1.0).   
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The preferential blockade of I_Ba,T by nociceptin is illustrated further by the current-voltage (I-V) curves shown in Figure 1C,D. The presence of I_Ba,T is seen as an inflection on the descending part of the curve between 50 and 25 mV (Fox et al., 1987). This inflection is attenuated in the presence of nociceptin (Fig. 1C) but not in the presence of morphine (Fig. 1D). At more positive voltages, N-, L-, P-, and presumptive Q-type conductances are activated (Fox et al., 1987; Rusin and Moises, 1995). Components of these HVA conductances were attenuated by both nociceptin and morphine. This is clear from the I-V curves shown in Figure 1C,D and from the data records in Figure 2A₁,B₁.

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Fig. 2. Effects of nociceptin and morphine on HVA g_Ba. A₁, B₁, Currents recorded in response to a step from 90 to 10 mV in the presence and absence of 1 µM nociceptin or 1 µM morphine. Note suppression of current and slowed rate of activation in the presence of agonists. A₂, B₂, Removal of suppression of HVA g_Ba induced by 1 µM nociceptin or 1 µM morphine with depolarizing prepulses to +100 mV. Current responses to depolarizing commands are off scale; 2 nA/20 msec calibration in A₁ refers also to records in B₁, and 2 nA/40 msec calibration in A₂ refers also to records in B₂. Records in A₁ and A₂ from the same cell and records in B₁ and B₂ from another cell.
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The EC₅₀ for nociceptin-induced suppression of I_Ba,T from the log-concentration-response relationship (Fig. 1E) was 100 nM with a Hill coefficient of 1.0 (Fig. 1F). Nociceptin (1 µM) completely blocked I_Ba,T in 22 of 77 cells tested. This contrasted with the actions of both nociceptin and morphine on HVA conductances, in which suppression rarely exceeded 70%.

Pharmacology of DRG T-current

The current that was inhibited by nociceptin was clearly I_Ba,T, because it was almost completely blocked by Ni²⁺ (100 µM) (DeWaard et al., 1996) yet was reduced by <25% by Cd²⁺ (100 µM; n = 4). By contrast, HVA conductances were selectively blocked by Cd²⁺ (100 µM) and were reduced by <25% by Ni²⁺ (100 µM; n = 4). In another experiment, we found that 20 µM Ni²⁺ produced clear suppression of I_Ba,T, with negligible effect on HVA I_Ba.

I_Ba,T was not affected by -conotoxin GVIA (1 µM; n = 3), and nifedipine (2 µM; n = 3) produced <10% suppression.

Ethosuximide, a drug that is used in the management of absence seizures, has been reported to inhibit I_Ba,T in thalamic relay (Coulter et al., 1990; Huguenard and Prince, 1994) and reticular neurons (Huguenard and Prince, 1994). This substance was without effect on I_Ba,T in DRG neurons (500 µM; n = 2).

Pharmacology of nociceptin-induced T-current suppression

Effects mediated via ORL₁ receptors are unaffected or relatively insensitive to inhibition by the broad spectrum µ-, -, and -opioid antagonist naloxone (Connor et al., 1996a,b; Faber et al., 1996). In agreement with this, naloxone (up to 1 µM) failed to antagonize the suppression of I_Ba,T by nociceptin (n = 3) (Fig. 3A₁,A₂). On the other hand, the ₃-agonist nalbzoh has been reported to exert an antagonist action at ORL₁ receptors (Dunnill et al., 1996). Figure 3B₂ shows that 1 µM nalbzoh almost completely prevented suppression of I_Ba,T by 1 µM nociceptin (n = 3) (Fig. 3B₁).

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Fig. 3. Pharmacology of nociceptin attenuation of T-current. Superimposed data records show (A₁) suppression of I_Ba,T by 1 µM nociceptin, (A₂) a second response to nociceptin recorded in the same cell in the presence of 1 µM naloxone, (B₁) suppression of I_Ba,T in another cell by 1 µM nociceptin, and (B₂) attenuation of nociceptin-induced inhibition in the second cell by 1 µM nalbzoh. I_Ba,T evoked by a step to 40 mV from holding potential of 90 mV (voltage record omitted for clarity).
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Nalbzoh also has significant antagonist action at µ-opioid receptors (Dunnill et al., 1996), and we found that it antagonized the actions of both morphine and nociceptin on HVA I_Ba (n = 4).

Role of G-proteins

Agonist-modulation of N-type Ca²⁺ channel currents via G-proteins is associated with slowing of the activation kinetics of the current as the channels are changed from a "willing" to a "reluctant" state (Bean, 1989; Dolphin, 1996). This type of kinetic slowing has been reported for the action of nociceptin on HVA I_Ca in hippocampal neurons (Knoflach et al., 1996). An obvious increase in the time constant for activation of HVA I_Ba is seen in the presence of morphine or nociceptin in Figure 2A₁,B₁. Suppression of HVA g_Ba was relieved by 75 msec depolarizing prepulses to +100 mV (Fig. 2A₂,B₂). This type of facilitation, which is also characteristic of G-protein-coupled responses (Dolphin, 1996), was reported previously by Womack and McCleskey (1995) for the action of the µ-opioid selective agonist PLO 17 (Tyr-Pro-N-Me-Phe-D-Pro-NH₂) on HVA I_Ba in DRG.

The inactivation kinetics of g_Ba,T prevented us from testing whether nociceptin suppression was relieved by a large depolarizing prepulse. There was no alteration, however, in the rate of activation of g_Ba,T when it was recorded in the presence of nociceptin. Figure 4A₁ illustrates moderate suppression of I_Ba,T by 100 nM nociceptin. The data records were normalized to the same amplitude and replotted in Figure 4A₂. The two records superimpose exactly, indicating that the rate of onset of the current, as well as the rate of inactivation at 40 mV, is unchanged by nociceptin. This raised the possibility that its actions involve a novel, G-protein-independent mechanism.

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Fig. 4. Evidence against G-protein involvement in the effect of nociceptin on T-currents. A₁, Superimposed data records to show suppression of I_Ba,T by 100 nM nociceptin. A₂, Superimposed records from A₁ normalized and replotted. Note that the two records superimpose exactly, and the presence of nociceptin does not slow the onset or the rate of inactivation of the current. B₁, Graph of time course of an experiment done with a pipette containing 100 µM GTP--S. Points represent amplitudes of I_Ba,T or HVA I_Ba evoked by a double-pulse protocol applied once every 20 sec. Both I_Ba,T and HVA I_Ba are suppressed by 1 µM nociceptin. Although I_Ba,T returns to control amplitude after nociceptin washout, HVA I_Ba remains suppressed throughout the course of the experiment. The cell had been left to stabilize with intracellularly applied GTP--S for 15 min before the application of nociceptin. B₂, Original superimposed data record collected before, during, and after application of nociceptin in the experiment plotted in B₁. The cell was stepped from 90 to 40 mV to activate g_Ba,T and then to 10 mV to activate HVA g_Ba. Nociceptin invokes reversible suppression of the former and irreversible suppression of the latter.
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This possibility was tested by substituting 100 µM GTP--S for the GTP that was normally included in the pipette solution. If the action of nociceptin on I_Ba,T did not involve a G-protein, it should produce reversible suppression of the current in the presence of GTP--S (Moises et al., 1994a). This seemed to be the case (n = 5). We took advantage of the use of HVA I_Ba as an internal positive control to demonstrate inhibition of G-protein inactivation by GTP--S. A double-step protocol from 90 mV was used to activate g_Ba,T at 40 mV and then to activate HVA g_Ba at 10 mV. Figure 4B₁ shows data from a cell studied with 100 µM GTP--S. The amplitudes of I_Ba,T responses and HVA I_Ba responses evoked once every 20 sec are plotted against time. Although the nociceptin-induced suppression of HVA g_Ba was irreversible, as would be expected for a G-protein-coupled response, complete recovery of nociceptin suppression of I_Ba,T was observed. Superimposed data records obtained before, during, and after nociceptin application are shown in Figure 4B₂. I_Ba,T recovers after nociceptin has been washed out, but HVA I_Ba does not. It may be noticed that nociceptin does not seem to invoke kinetic slowing of HVA I_Ba in Figure 4B₂. This may reflect the presence of GTP--S in the patch pipette.

GDP--S irreversibly inactivates G-proteins (Burch and Axelrod, 1987). As might be expected, replacement of GTP in the patch pipette with 2 mM GDP--S prevented modulation of HVA g_Ba by nociceptin. I_Ba,T suppression in the same cell, at the same time, was unaffected (n = 4). A typical experiment, using the double-pulse protocol to sequentially activate g_Ba,T and HVA g_Ba, is illustrated in Figure 5A. The superimposed records show that nociceptin suppresses I_Ba,T but has no effect on HVA conductances.

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Fig. 5. Further evidence against G-protein involvement in the effect of nociceptin on T-currents. A, Effect of 1 µM nociceptin on I_Ba,T and HVA I_Ba in the same cell studied with 2 mM GDP--S in the pipette. The cell had been allowed to equilibrate with intracellularly applied GDP--S for 15 min before the application of nociceptin. Superimposed records acquired before and during nociceptin application show that I_Ba,T was suppressed when HVA I_Ba was unaffected. B, An experiment similar to that of A but using AlF₃ (100 µM) instead of GDP--S in the pipette. The cell had been allowed to equilibrate with intracellularly applied AlF₃ for 20 min before the application of 1 µM nociceptin. Superimposed records acquired before, during, and after nociceptin application show that I_Ba,T was reversibly suppressed when HVA I_Ba was unaffected. Voltage protocol in B applies also to experiment illustrated in A.
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G-protein function can also be modified radically by AlF₃. This substance acts as the AlF₄ anion, which is an analog of the -phosphate of GTP (Bigay et al., 1987). AlF₃ therefore strongly activates G-proteins and thereby occludes the response of their downstream targets to agonists. In our experiments, HVA g_Ba decreased to ~20% of control after ~20 min when 100 µM AlF₃ was included in the patch pipette. I_Ba,T was relatively stable under these conditions and was still susceptible to reversible suppression by nociceptin (n = 4). A typical experiment is illustrated in Figure 5B. The superimposed records show that nociceptin reversibly suppresses I_Ba,T when HVA conductances in the same cell are refractory to modulation by G-protein-coupled agonists.

Biophysical aspects of the action of nociceptin

Inhibition of I_Ba,T by nociceptin did not exhibit "use-dependence." Because the time constant for removal of I_Ba,T inactivation at 90 mV is ~1 sec (Carbone and Lux, 1987), we were unable to repeatedly activate the current, even with 30 msec pulses, at frequencies above 0.4 Hz. These brief pulses were used to limit the amount of inactivation that developed at 40 mV, so that less inactivation had to be removed between the pulses at 90 mV. When nociceptin was applied while I_Ba,T was repeatedly evoked at 0.4 Hz, the current attained a reduced steady-state amplitude within 3 min of initiating nociceptin superfusion. When nociceptin was washed out and reapplied in the absence of channel opening, a similar rate and extent of depression of I_Ba,T was seen (n = 4). The block was therefore not use-dependent. In the experiment illustrated in Figure 6A, the downward deflections are low-speed chart recordings of successive current responses (I_Ba,T) to 30 msec pulses to 40 mV (from V_h = 90 mV) delivered at 0.4 Hz. The brief I_Ba,T responses and the associated tail and capacitative currents cannot be distinguished on the chart. The progressive decrease in amplitude of the deflections reflects suppression of the current by nociceptin. When nociceptin is reapplied in the absence of repeated I_Ba,T responses, the same amount of block still occurs at about the same rate.

In other experiments, in which both I_Ba,T and HVA I_Ba were recorded, there seemed to be a slight delay in the onset of block of HVA I_Ba compared with I_Ba,T.

We were also able to exclude an alteration in steady-state inactivation as the mechanism for a nociceptin-induced decrease in I_Ba,T. Figure 6B shows the steady-state inactivation curve for I_Ba,T; the current was evoked at 40 mV following steps from various prepulse potentials. The voltage protocol is shown as an inset to the graph. Maximal I_Ba,T was evoked from holding potentials exceeding 105 mV, and currents evoked from different prepulse potentials were normalized to this maximal value. In the presence of 100 nM nociceptin, which reduced I_Ba,T recorded at 40mV by 36%, there was no obvious change in the normalized steady-state inactivation curve. Original data records are shown in Figure 5C₁,C₂. Similar effects were seen in a total of five cells.

Effect of nociceptin on action potentials and burst discharges

I_Ca,T plays a role in the production of burst discharges in various neuronal types (Huguenard, 1996), and it has been reported to underlie an afterdepolarization that follows a single AP in some DRG neurons (White et al., 1989). This afterdepolarization gives rise to a burst of two or more successive APs in response to a single brief stimulus. Typical current-clamp recordings from a cell of this type are shown in Figure 7. The afterdepolarization and associated second and third spikes were attenuated by 1 µM nociceptin (n = 4) (Fig. 7A) but not by 1 µM morphine (n = 4) (Fig. 7B). Figure 7C illustrates suppression of the afterdepolarization by Ni²⁺ (100 µM; n = 4), a result consistent with the involvement of I_Ca,T in its generation (White et al., 1989).

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Fig. 7. Effects of nociceptin, morphine, and Ni²⁺ on APs, bursting activity, and afterdepolarizations. All records were obtained from the same cell. APs were evoked from a 5 msec/0.3 nA depolarizing current command applied via the recording electrode. Superimposed records of bursts of APs were recorded before and during superfusion of (A) 1 µM nociceptin, (B) 1 µM morphine, and (C) 100 µM Ni²⁺. Note that afterdepolarization and burst of spikes are attenuated by nociceptin and Ni²⁺ but not by morphine. Calibration (40 mV/40 msec) in B and current trace in C apply to all records.
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DISCUSSION

We find that the novel opioid peptide nociceptin selectively inhibits I_Ba,T in medium-sized DRG neurons by a G-protein-independent mechanism. This effect is reflected in impairment of the ability of the neurons to generate bursts of APs in response to a brief excitatory stimulus.

Unlike HVA g_Ba, which is subject to regulation by a wide variety of neuromodulators that act through heptahelical, G-protein-coupled receptors (Dolphin, 1996), very few agonists have been reported to modulate I_Ba,T (Kobrinsky et al., 1994; Huguenard, 1996). Although I_Ba,T in sensory neurons is decreased when G-proteins are activated with GTP--S (Gross et al., 1990), this mechanism does not seem to be involved in the action of nociceptin. Because the G-protein-mediated effects of opioid agonists on HVA g_Ba are well characterized in DRG neurons (Moises et al., 1994a,b; Wilding et al., 1995; Womack and McCleskey, 1995), each cell that was studied served as its own control. This allowed us to convincingly exclude a role for G-proteins, because nociceptin attenuated I_Ba,T after its effects on HVA g_Ba were compromised with GDP--S or AlF₃ (Fig. 5). Similarly, in cells in which G-protein-coupled effects of nociceptin on HVA g_Ba were rendered irreversible by the inclusion of GTP--S in the pipette, nociceptin invoked a reversible suppression of I_Ba,T (Fig. 4B). The fact that nociceptin clearly altered the activation kinetics of HVA g_Ba (Fig. 2A) yet failed to alter that of g_Ba,T (Fig. 4A) may also be consistent with modulation of T-type Ca²⁺ channels by a G-protein-independent mechanism (but see Kobrinsky et al., 1994).

Nociceptin was identified as an endogenous opioid agonist for the heptahelical ORL₁ receptor (Meunier et al., 1995; Reinscheid et al., 1995; Nothacker et al., 1996), and Connor et al. (1996b) showed that its effects on Ca²⁺ channel currents and intracellular Ca²⁺ accumulation in neuroblastoma cells are blocked by pertussis toxin. Because the effects of nociceptin on I_Ba,T in DRG neurons do not seem to involve G-proteins, this raises the possibility that they may not be mediated via the heptahelical ORL₁ receptor. Although the effect of nociceptin is antagonized by nalbzoh (Fig. 2B) (Dunnill et al., 1996), the selectivity of this antagonist is yet to be established. On the other hand, the EC₅₀ for the action of nociceptin on I_Ba,T of 100 nM is comparable with that for its presumed actions at ORL₁ receptors (22-90 nM) (Connor et al., 1996a,b; Vaughan and Christie, 1996; Vaughan et al., 1997). In our opinion, neither the effect of nalbzoh nor the similarities in EC₅₀ values allow the unequivocal implication or exclusion of a role for ORL₁ in the actions of nociceptin on I_Ba,T. One remote possibility is that the activated ORL₁ receptor interacts directly with the T-type Ca²⁺ channel without the intervention of a G-protein.

Schroeder et al. (1991) reported that g_Ca,T in sensory neurons is attenuated by the µ-receptor selective agonist D-Ala², N-Me-Phe⁴-Gly⁵-ol enkephalin (DAMGO). It is therefore possible that only certain opioid peptides attenuate I_Ba,T and that this action is not shared by other opioids, including PLO17 (Moises et al., 1994b) and morphine. It therefore would be of interest to determine whether the action of DAMGO on T-channel currents described by Schroeder et al. (1991) is G-protein-mediated and whether it is blocked by naloxone. This antagonist was ineffective in antagonizing the action of nociceptin on I_Ba,T (Fig. 2A). Two other reports of effects of opioid peptides that do not seem to involve G-proteins have already appeared. Twitchell and Rane (1994) were unable to implicate G-proteins in the potentiation of Ca²⁺-sensitive K⁺ current by the µ-selective agonist DAMGO, and Brauneis et al. (1996) showed that dynorphin inhibited currents through recombinant NMDA channels expressed in Xenopus oocytes that did not express opioid receptors. Perhaps G-protein-independent actions contribute to behavioral effects seen with opioid peptides in whole animals. Thus, suppression of I_Ca,T by nociceptin may contribute to the hyperalgesic effect seen in tail-flick tests when it is administered intracerebroventricularly to mice (Meunier et al., 1995; Reinscheid et al., 1995).

Little can be said, at this stage, about the molecular mechanism of the action of nociceptin. Because effects on steady-state inactivation and effects mediated via G-proteins have been excluded, nociceptin may interact directly with the T-type Ca²⁺ channel. Because the EC₅₀ is 100 nM, this would predict a residency time (T) of ~100 msec on its receptor site. This was estimated by assuming a K_D of 10⁷ that is equal to the EC₅₀ and using the approximation that T = 10⁸/K_D (Hille, 1992). A relatively long residency time suggests that nociceptin would not produce a "flickering" type of open-channel block, and this is consistent with the observation that it did not affect the rate of activation or inactivation of I_Ba,T (Fig. 4A₂). Direct interaction of nociceptin with T-type Ca²⁺ channels could therefore involve "closed-channel block," as has been proposed for ethosuximide block of thalamic channels (Coulter et al., 1991), or a very rapidly developing block of open channels in which the peptide would enter each channel as soon as it opens and prevent the passage of ions. In either case, no use-dependence would be observed.

Activation of a receptor tyrosine kinase is another plausible, G-protein-independent mechanism for the action of a peptide such as nociceptin. The possible relevance of this mechanism derives from the observation that HVA I_Ca in molluscan neurons is enhanced within minutes of application of mammalian nerve growth factor (Wildering et al., 1995). Also, peptides such as angiotensin II and vasopressin can act via tyrosine kinase pathways to cause contraction of certain types of smooth muscle (Hollenberg, 1994). A third possibility is that nociceptin exerts its effects via activation of membrane-bound guanylyl cyclase in the same way as atrial natriuretic factor (Fenrick et al., 1994). These and other possible mechanisms could be readily examined in future work; it may be that the preservation of a nociceptin response in the presence of GTP analogs and AlF₃ precludes a role for the direct activation of guanylyl cyclase.

The observation that I_Ba,T in DRG is blocked by nociceptin but not by ethosuximide may reflect differences in the properties of the T-type Ca²⁺ channels in thalamic neurons compared with sensory neurons. This goes along with the idea that several subtypes of T-channels exist in neurons (Kobrinsky et al., 1994; Huguenard, 1996).

Because I_Ca,T is responsible for generating repetitive or bursting discharges, for boosting Ca²⁺ entry, and for low-threshold spike generation (Huguenard, 1996), it can contribute to convulsant activity in central neurons. The medium-sized DRG neurons used in the present study also tend to exhibit short bursts of 2-4 APs in response to a brief depolarizing stimulus. This burst of APs originates from an afterdepolarization that follows the first AP. Our results (Fig. 7C) with the T-channel blocker Ni²⁺ (DeWaard et al., 1996) support the hypothesis that I_Ca,T underlies the generation of this afterdepolarization (White et al., 1989). Because nociceptin binding sites have been detected in various CNS regions, including cortex and thalamus (Sim et al., 1996), the observation that nociceptin blocks both I_Ba,T and bursting activity (Fig. 7) raises the possibility that it may be an endogenous anticonvulsant. Because nociceptin blocks I_Ba,T in a cell type in which ethosuximide is ineffective, the development of appropriate nociceptin analogs may provide a logical route for the development of new anticonvulsant therapies.

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FOOTNOTES

Received July 14, 1997; revised Sept. 2, 1997; accepted Sept. 3, 1997.

  

This work was supported by the Alberta Paraplegic Foundation, the Rick Hansen Man-in-Motion Foundation, and the Medical Research Council (MRC) of Canada. Dr. F. A. Abdulla gratefully acknowledges fellowship support from the Alberta Heritage Foundation for Medical Research. We thank Drs. F. Tse, W. F. Colmers, and A. Tse for useful discussions and for their comments on an early version of this manuscript.

Correspondence should be addressed to Dr. Peter A. Smith, Department of Pharmacology, University of Alberta, 9.75 Medical Sciences Building, Edmonton, Alberta, Canada, T6G 2H7.

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