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-, 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 (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-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-inducedI Ba,T suppression. By contrast, both morphine and nociceptin suppressed HVAg Ca, 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 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.
- LC132 receptor
- neuropathic pain
- sympathetic ganglion
- orphan receptor
- spinal cord
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 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 HVAg 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 HVAg Ca by a G-protein-dependent mechanism, only nociceptin affects the low-voltage-activated (LVA), transientI 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).
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 I Ca with Ba2+ as charge carrier (I Ba). 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.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 (τm) using the equation τm= C in R in. Under voltage clamp, C in 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 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 andC 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 andC 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-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 6 A, were produced using Origin 4.10′ (Microcal).
Suppression of T-current by nociceptin
Nociceptin (0.01–1.0 μm) suppressedI Ba,T in all cells tested (n > 100). Figure1 A1 illustrates almost complete suppression of I Ba,Tby 1 μm nociceptin in a medium-sized DRG neuron. An equal concentration of morphine produced only a small (<10%) suppression ofI Ba,T in the same cell (Fig.1 A2 ). Morphine (up to 10 μm) produced no more than 20% suppression ofI 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. 1 B).
The preferential blockade of I Ba,T by nociceptin is illustrated further by the current–voltage (I–V) curves shown in Figure 1 C,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. 1 C) but not in the presence of morphine (Fig.1 D). 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 1 C,D and from the data records in Figure2 A1 ,B1 .
The EC50 for nociceptin-induced suppression ofI Ba,T from the log-concentration-response relationship (Fig. 1 E) was 100 nm with a Hill coefficient of 1.0 (Fig. 1 F). 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 clearlyI 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 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 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-, 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 I Ba,T by nociceptin (n = 3) (Fig.3 A1 ,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 3 B2 shows that 1 μm nalbzoh almost completely prevented suppression ofI Ba,T by 1 μm nociceptin (n = 3) (Fig. 3 B1 ).
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 Figure2 A1 ,B1 . Suppression of HVA g Ba was relieved by 75 msec depolarizing prepulses to +100 mV (Fig.2 A2 ,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 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. Figure4 A1 illustrates moderate suppression of I Ba,T by 100 nm nociceptin. The data records were normalized to the same amplitude and replotted in Figure 4 A2 . 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.
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 I Ba,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 HVAI Ba 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 activateg Ba,T at −40 mV and then to activate HVAg Ba at −10 mV. Figure4 B1 shows data from a cell studied with 100 μm GTP-γ-S. The amplitudes ofI Ba,T responses and HVAI 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 4 B2 .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 HVAI Ba in Figure 4 B2 . 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 HVAg Ba by nociceptin. I Ba,Tsuppression 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 HVAg Ba, is illustrated in Figure5 A. The superimposed records show that nociceptin suppresses I Ba,T but has no effect on HVA conductances.
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 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 5 B. 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 ofI 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 whileI 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 Figure6 A, 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 Bacompared with I Ba,T.
We were also able to exclude an alteration in steady-state inactivation as the mechanism for a nociceptin-induced decrease inI Ba,T. Figure 6 B shows the steady-state inactivation curve forI 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,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 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 Figure5 C1 ,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 Figure7. The afterdepolarization and associated second and third spikes were attenuated by 1 μmnociceptin (n = 4) (Fig. 7 A) but not by 1 μm morphine (n = 4) (Fig. 7 B). Figure 7 C illustrates suppression of the afterdepolarization by Ni2+ (100 μm; n = 4), a result consistent with the involvement ofI Ca,T in its generation (White et al., 1989).
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). AlthoughI Ba,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 HVAg 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-β-S or AlF3 (Fig. 5). Similarly, in cells in which G-protein-coupled effects of nociceptin on HVAg Ba were rendered irreversible by the inclusion of GTP-γ-S in the pipette, nociceptin invoked a reversiblesuppression of I Ba,T (Fig.4 B). The fact that nociceptin clearly altered the activation kinetics of HVA g Ba (Fig.2 A) yet failed to alter that ofg Ba,T (Fig. 4 A) 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.2 B) (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 onI 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 agonistd-Ala2,N-Me-Phe4-Gly5-ol enkephalin (DAMGO). It is therefore possible that only certain opioidpeptides 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.2 A). 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 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 10−7 that is equal to the EC50 and using the approximation that T = 10−8/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.4 A2 ). 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. 7 C) 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.
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.