Articles

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

Fuad A. Abdulla and Peter A. Smith
Journal of Neuroscience 15 November 1997, 17 (22) 8721-8728; https://doi.org/10.1523/JNEUROSCI.17-22-08721.1997

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 gCa) in much the same way as μ-, δ-, and κ-opioids. We report an action of nociceptin that is not shared by morphine: the suppression of low-voltage-activated, transient calcium (barium) current (IBa,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-γ-S (100 μm), GDP-β-S (2 mm), or aluminum fluoride (AlF3) (100 μm) in the patch pipette failed to provide evidence for G-protein involvement in nociceptin-inducedIBa,T suppression. By contrast, both morphine and nociceptin suppressed HVAgCa, and the latter response was affected by intracellular GTP-γ-S, GDP-β-S, and AlF3 in ways that confirmed G-protein involvement. The selective effect of nociceptin on IBa,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.

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-, δ-, and κ-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-, δ-, or κ-opioids attenuate high-voltage-activated Ca2+channel conductances (HVA gCa) (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 (gKIR) (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 HVAgCa in hippocampal neurons (Knoflach et al., 1996) and in a neuroblastoma cell line (Connor et al., 1996b). Nociceptin activates gKIR 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 gCa 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 HVAgCa by a G-protein-dependent mechanism, only nociceptin affects the low-voltage-activated (LVA), transientICa (T-current,ICa,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 ICa,N is normally associated with neurotransmitter release (DeWaard et al., 1996), ICa,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 ICa,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).

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 byWhite 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 ICa with Ba2+ as charge carrier (IBa). Borosilicate glass patch electrodes had DC resistances of 4–6 MΩ for AP recording or 1–3 MΩ 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.IBa 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 (gCl,Ca) (Mayer, 1985) were not observed. A P/4 leak subtraction paradigm was used when necessary.

Under bridge-balance current-clamp, input capacitance (Cin) was calculated from the input resistance (Rin) and the membrane time constant (τm) using the equation τm= CinRin. Under voltage clamp, Cin was measured by integrating the area of capacitative current transients that were generated by 10 mV commands (ΔV). This yielded the charge Q that is related to Cin by Q =VCin. 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 andCin >90 pF; “medium” cells had an AP duration of 3–5 msec and Cin of 70–90 pF; and “small” cells had an AP duration >5 msec andCin <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 (IBa,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 IBa,T. Only cells with “giant”IBa,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 IBa, 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,IBa,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 (Vh) 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-γ-S; trilithium salt) were from Research Biochemical International (Natick, MA), and guanosine-5′-O-(2-thiodiphosphate) (GDP-β-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).

Fig. 6.
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 gBa,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 (atleft) while gBa,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 ofgBa,T activation. Voltage commands were recommenced 3 min after initiating nociceptin superfusion, andIBa,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 forgBa,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 gBa,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 IBa,T.

RESULTS

Suppression of T-current by nociceptin

Nociceptin (0.01–1.0 μm) suppressedIBa,T in all cells tested (n > 100). Figure1A1 illustrates almost complete suppression of IBa,Tby 1 μm nociceptin in a medium-sized DRG neuron. An equal concentration of morphine produced only a small (<10%) suppression ofIBa,T in the same cell (Fig.1A2 ). Morphine (up to 10 μm) produced no more than 20% suppression ofIBa,T in 18 of 67 cells tested, and the remainder (49) were unaffected. Nociceptin-induced inhibition was reflected in attenuation of IBa,T tails recorded at −60 mV (Fig. 1B).

Fig. 1.
Fig. 1.

Blockade of IBa,T by nociceptin. A1 , A2 , Comparison of the effects of nociceptin and morphine onIBa,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 IBa,T by 1 μmnociceptin. A2 , In the same cell, 1 μm morphine suppresses the current by <10%.B, Superimposed records obtained from another cell to show suppression of IBa,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–Vrelationships for IBa recorded in the absence and presence of 1 μm nociceptin.Asterisk marks characteristic shoulder onI–V relationship that results fromIBa,T. Note attenuation of current at −40 mV (IBa,T) and −10 mV (attenuation of HVA IBa) by nociceptin.D, I–V relationships forIBa recorded in the absence and presence of 1 μm morphine. Note lack of attenuation of current at potentials more negative than −40 mV (IBa,T, marked byasterisk) and attenuation of HVAIBa 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 IBa,T(EC50) is 100 nm. F, Data from E replotted as a Hill plot [Log10R/R −Rm vs Log10 nociceptin concentration ÷ EC50; where Ris amplitude of response and Rm is the maximum response that can be obtained;KD, 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).  

The preferential blockade of IBa,T by nociceptin is illustrated further by the current–voltage (I–V) curves shown in Figure 1C,D. The presence of IBa,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–Vcurves shown in Figure 1C,D and from the data records in Figure2A1 ,B1 .

Fig. 2.
Fig. 2.

Effects of nociceptin and morphine on HVAgBa. A1 ,B1 , Currents recorded in response to a step from −90 to −10 mV in the presence and absence of 1 μmnociceptin 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 gBa 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 inA1 refers also to records inB1, and 2 nA/40 msec calibration inA2 refers also to records inB2. Records in A1and A2 from the same cell and records inB1 and B2 from another cell.

The EC50 for nociceptin-induced suppression ofIBa,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 IBa,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 clearlyIBa,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 IBa,T, with negligible effect on HVA IBa.

IBa,T was not affected by ω-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 IBa,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 IBa,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-, δ-, and κ-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 IBa,T by nociceptin (n = 3) (Fig.3A1 ,A2 ). On the other hand, the κ3-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 ofIBa,T by 1 μm nociceptin (n = 3) (Fig. 3B1 ).

Fig. 3.
Fig. 3.

Pharmacology of nociceptin attenuation of T-current. Superimposed data records show (A1) suppression ofIBa,T by 1 μm nociceptin, (A2) a second response to nociceptin recorded in the same cell in the presence of 1 μmnaloxone, (B1) suppression ofIBa,T in another cell by 1 μmnociceptin, and (B2) attenuation of nociceptin-induced inhibition in the second cell by 1 μmnalbzoh. IBa,T evoked by a step to −40 mV from holding potential of −90 mV (voltage record omitted for clarity).

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 IBa(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 ICa in hippocampal neurons (Knoflach et al., 1996). An obvious increase in the time constant for activation of HVA IBa is seen in the presence of morphine or nociceptin in Figure2A1 ,B1 . Suppression of HVA gBa 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 byWomack and McCleskey (1995) for the action of the μ-opioid selective agonist PLO 17 (Tyr-Pro-N-Me-Phe-d-Pro-NH2) on HVA IBa in DRG.

The inactivation kinetics of gBa,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 gBa,T when it was recorded in the presence of nociceptin. Figure4A1 illustrates moderate suppression of IBa,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.

Fig. 4.
Fig. 4.

Evidence against G-protein involvement in the effect of nociceptin on T-currents.A1, Superimposed data records to show suppression of IBa,T by 100 nmnociceptin. 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 μmGTP-γ-S. Points represent amplitudes ofIBa,T or HVA IBaevoked by a double-pulse protocol applied once every 20 sec. BothIBa,T and HVA IBaare suppressed by 1 μm nociceptin. AlthoughIBa,T returns to control amplitude after nociceptin washout, HVA IBa remains suppressed throughout the course of the experiment. The cell had been left to stabilize with intracellularly applied GTP-γ-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 activategBa,T and then to −10 mV to activate HVAgBa. Nociceptin invokes reversible suppression of the former and irreversible suppression of the latter.

This possibility was tested by substituting 100 μmGTP-γ-S for the GTP that was normally included in the pipette solution. If the action of nociceptin on IBa,Tdid not involve a G-protein, it should producereversible suppression of the current in the presence of GTP-γ-S (Moises et al., 1994a). This seemed to be the case (n = 5). We took advantage of the use of HVAIBa as an internal positive control to demonstrate inhibition of G-protein inactivation by GTP-γ-S. A double-step protocol from −90 mV was used to activategBa,T at −40 mV and then to activate HVAgBa at −10 mV. Figure4B1 shows data from a cell studied with 100 μm GTP-γ-S. The amplitudes ofIBa,T responses and HVAIBa responses evoked once every 20 sec are plotted against time. Although the nociceptin-induced suppression of HVA gBa was irreversible, as would be expected for a G-protein-coupled response, complete recovery of nociceptin suppression of IBa,T was observed. Superimposed data records obtained before, during, and after nociceptin application are shown in Figure 4B2 .IBa,T recovers after nociceptin has been washed out, but HVA IBa does not. It may be noticed that nociceptin does not seem to invoke kinetic slowing of HVAIBa in Figure 4B2 . This may reflect the presence of GTP-γ-S in the patch pipette.

GDP-β-S irreversibly inactivates G-proteins (Burch and Axelrod, 1987). As might be expected, replacement of GTP in the patch pipette with 2 mm GDP-β-S prevented modulation of HVAgBa by nociceptin. IBa,Tsuppression in the same cell, at the same time, was unaffected (n = 4). A typical experiment, using the double-pulse protocol to sequentially activate gBa,T and HVAgBa, is illustrated in Figure5A. The superimposed records show that nociceptin suppresses IBa,T but has no effect on HVA conductances.

Fig. 5.
Fig. 5.

Further evidence against G-protein involvement in the effect of nociceptin on T-currents. A, Effect of 1 μm nociceptin on IBa,T and HVAIBa in the same cell studied with 2 mm GDP-β-S in the pipette. The cell had been allowed to equilibrate with intracellularly applied GDP-β-S for 15 min before the application of nociceptin. Superimposed records acquired before and during nociceptin application show thatIBa,T was suppressed when HVAIBa was unaffected. B, An experiment similar to that of A but using AlF3 (100 μm) instead of GDP-β-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 thatIBa,T was reversibly suppressed when HVAIBa was unaffected. Voltage protocol inB applies also to experiment illustrated inA.

G-protein function can also be modified radically by AlF3. This substance acts as the AlF4− anion, which is an analog of the γ-phosphate of GTP (Bigay et al., 1987). AlF3therefore strongly activates G-proteins and thereby occludes the response of their downstream targets to agonists. In our experiments, HVA gBa decreased to ∼20% of control after ∼20 min when 100 μm AlF3 was included in the patch pipette. IBa,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 IBa,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 IBa,T by nociceptin did not exhibit “use-dependence.” Because the time constant for removal ofIBa,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 whileIBa,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 IBa,T was seen (n = 4). The block was therefore not use-dependent. In the experiment illustrated in Figure6A, the downward deflections are low-speed chart recordings of successive current responses (IBa,T) to 30 msec pulses to −40 mV (from Vh = −90 mV) delivered at 0.4 Hz. The brief IBa,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 IBa,T responses, the same amount of block still occurs at about the same rate.

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

We were also able to exclude an alteration in steady-state inactivation as the mechanism for a nociceptin-induced decrease inIBa,T. Figure 6B shows the steady-state inactivation curve forIBa,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 IBa,Twas 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 IBa,T recorded at 40mV by 36%, there was no obvious change in the normalized steady-state inactivation curve. Original data records are shown in Figure5C1 ,C2 . Similar effects were seen in a total of five cells.

Effect of nociceptin on action potentials and burst discharges

ICa,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 Figure7. The afterdepolarization and associated second and third spikes were attenuated by 1 μmnociceptin (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 ofICa,T in its generation (White et al., 1989).

Fig. 7.
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.

DISCUSSION

We find that the novel opioid peptide nociceptin selectively inhibits IBa,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 gBa, 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 IBa,T(Kobrinsky et al., 1994; Huguenard, 1996). AlthoughIBa,T in sensory neurons is decreased when G-proteins are activated with GTP-γ-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 HVAgBa 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 IBa,T after its effects on HVA gBa were compromised with GDP-β-S or AlF3 (Fig. 5). Similarly, in cells in which G-protein-coupled effects of nociceptin on HVAgBa were rendered irreversible by the inclusion of GTP-γ-S in the pipette, nociceptin invoked a reversiblesuppression of IBa,T (Fig.4B). The fact that nociceptin clearly altered the activation kinetics of HVA gBa (Fig.2A) yet failed to alter that ofgBa,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 IBa,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 onIBa,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 IBa,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 gCa,T in sensory neurons is attenuated by the μ-receptor selective agonistd-Ala2,N-Me-Phe4-Gly5-ol enkephalin (DAMGO). It is therefore possible that only certain opioidpeptides attenuate IBa,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 IBa,T (Fig.2A). Two other reports of effects of opioidpeptides 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, andBrauneis 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 peptidesin whole animals. Thus, suppression of ICa,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 KD of 10−7 that is equal to the EC50 and using the approximation that T = 10−8/KD (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 IBa,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 ICa 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 IBa,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 ICa,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 ICa,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 IBa,T and bursting activity (Fig. 7) raises the possibility that it may be an endogenous anticonvulsant. Because nociceptin blocks IBa,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.

Footnotes

  • 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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Nociceptin Inhibits T-Type Ca2+ Channel Current in Rat Sensory Neurons by a G-Protein-Independent Mechanism
Fuad A. Abdulla, Peter A. Smith
Journal of Neuroscience 15 November 1997, 17 (22) 8721-8728; DOI: 10.1523/JNEUROSCI.17-22-08721.1997
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