Studies have shown that nociceptin, the endogenous ligand for the opioid receptor-like receptor (ORL1), modulates central control of cardiovascular activity. The nucleus ambiguus, an area containing cardiac parasympathetic neurons, contains both ORL1 receptors and neurons that contain nociceptin itself. Although previous work has shown that nociceptin acts to increase parasympathetic outflow to the heart, the mechanisms by which this is achieved are unknown. In the present study, the effects of nociceptin on spontaneous γ-aminobutyric acidergic (GABAergic) input to cardiac parasympathetic neurons (IPSCs) was examined. At 100 μM, nociceptin inhibited both the frequency (−35.6%) and the amplitude (−49.5%) of spontaneous GABAergic IPSCs in cardiac vagal neurons. Nociceptin also caused a novel postsynaptic inhibition of the responses evoked by exogenous application of GABA. These results indicate that nociceptin acts both on neurons precedent to cardiovascular neurons to decrease the activity of GABAergic neurons that synapse upon cardiovascular neurons and directly, inhibiting the postsynaptic currents evoked by GABA. This inhibition by nociceptin would increase parasympathetic outflow to the heart, thus providing a possible mechanism for nociceptin-induced bradycardia.
Nociceptin, a newly discovered endogenous peptide, is the ligand for the opioid receptor-like receptor (ORL1) (Henderson and McKnight, 1997; Meunier, 1997). Despite its selective binding to this receptor, nociceptin shares a structural resemblance to classical endogenous opioid peptides and displays actions in common with them, including modulation of pain (Henderson and McKnight, 1997; King et al., 1997) and cellular actions such as inhibition of synaptic transmission (Emmerson and Miller, 1999); regulation of neurotransmitter release including serotonin (Siniscalchi et al., 1999;Schlicker and Morari, 2000), noradrenaline (Schlicker and Morari, 2000;Trendelenburg et al., 2000), glutamate (Faber et al., 1996), dopamine (Murphy et al., 1996), and acetylcholine (Patel et al., 1997); and modulation of calcium conductances (Knoflach et al., 1996; Abdulla and Smith, 1997; Connor and Christie, 1998). In addition, nociceptin has been implicated in the modulation of cardiovascular activity. It has been shown to decrease cardiac output and total peripheral resistance (Champion et al., 1997), and to produce hypotension (Champion and Kadowitz 1997a,b; Giuliani et al., 1997; Salis et al., 2000) and bradycardia (Giuliani et al., 1997; Kapusta, 2000; Salis et al., 2000) in anesthetized rats; these effects were resistant to blockade with the classic opioid antagonist naloxone (Champion and Kadowitz, 1997b), indicating the probable stimulation of ORL1receptors. Using anesthetized rats, microinjection of nociceptin into the rostral ventrolateral medulla, one area of the brain involved in central control of cardiovascular activity, induces the same hypotensive and bradycardic responses as those induced by systemic administration of the drug (Chu et al., 1998, 1999a; Kapusta et al., 1999; Kapusta, 2000). Addition of nociceptin caused a profound inhibition of electrical activity of rostral ventrolateral medulla neurons in rat brain slices (Chu et al., 1998, 1999b), as well as those from other regions of the brain thought to be involved in control of cardiovascular function, including hypothalamic paraventricular neurons (Shirasaka et al., 2001). The nucleus tractus solitarius (NTS), a medullary area where cardiac sensory afferents terminate, has a rich distribution of ORL1 receptors (Lawrence and Jarrott, 1996), which probably mediate the inhibitory effect of nociceptin on baroreflex bradycardia seen with microinjection of nociceptin into this area (Lawrence and Jarrott, 1996; Mao and Wang, 2000).
A functionally important pathway from the NTS is to the nucleus ambiguus, an area within the medulla containing, among others, preganglionic parasympathetic cardiac neurons (Spyer and Gilbey, 1988). The axons of parasympathetic cardiac neurons descend in the vagus nerves and synapse upon neurons in cardiac ganglia located at the base of the heart. Most parasympathetic activity regulating heart rate and cardiac function originates from central parasympathetic cardiac neurons within the nucleus ambiguus (Mendelowitz, 1999; Wang et al., 2001a,b). Recent studies have revealed two major synaptic inputs to cardiac vagal neurons from the NTS: an excitatory glutamatergic pathway (Mendelowitz, 1998; Neff et al., 1998b) and an inhibitory GABAergic input (DiMicco et al., 1979; Wang et al., 2001a,b). Nicotine has also been found to facilitate glutamatergic transmission, as well as to directly activate vagal cardioinhibitory neurons (Neff et al., 1998a). However, little is known regarding modulation of GABAergic neurotransmission to cardiac vagal neurons. Studies utilizing techniques such as agonist-stimulated ([35S]GTPγS) binding (Sim and Childers, 1997), in situ hybridization, and immunohistochemistry (Neal et al., 1999a,b; Houtani et al., 2000; Mollereau and Mouledous, 2000) have identified both ORL1 receptors (Sim and Childers, 1997; Neal et al., 1999a; Houtani et al., 2000; Mollereau and Mouledous, 2000) and nociceptin itself (Neal et al., 1999b) within the NTS and nucleus ambiguus. Despite this, the effects of nociceptin on cardiac vagal neurons within the nucleus ambiguus remain unknown. This study therefore explored the effects of nociceptin on GABAergic innervation of cardiac vagal neurons in vitro using whole-cell patch-clamp recordings.
Materials and Methods
In an initial surgery, Sprague-Dawley rats (postnatal, days 6–10) were anesthetized by methoxyflurane and hypothermia, and they received a right thoracotomy. The heart was exposed and rhodamine (XRITC; Molecular Probes, Eugene, OR) was injected into the pericardial sac to antidromically label cardiac vagal neurons. On the day of the experiment (2–4 days later), the animals were anesthetized by methoxyflurane and hypothermia and killed by rapid cervical dislocation. The hindbrain was rapidly removed and placed in cold (0–2°C) buffer of the following composition: 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 25 mM glucose, 10 mM HEPES, and oxygenated with 100% O2. Slices of medulla 350 μm thick were cut using a Vibratome. These were mounted in a perfusion chamber and submerged in perfusate of the following composition: 128 mM NaCl, 3 mM KCl, 1.5 mM CaCl2, 1 mM MgCl2, 24 mM NaHCO3, 0.5 mM NaH2PO4, 30 mM glucose, oxygenated with a 95% O2/5% CO2 gas mixture. The osmolarity of both solutions was 285 to 290 mOsm and the pH was maintained between 7.35 and 7.4.
Individual cardiac neurons were identified by the presence of the fluorescent tracer (Mendelowitz and Kunze, 1991) and imaged with differential contrast optics, infrared illumination, and infrared-sensitive video detection cameras to visually guide and position the patch pipette onto the surface of the identified neuron. Pipettes were made with a puller (Narishige, Tokyo, Japan); filled resistances were 2 to 4 MΩ in the bath. The electrode solution contained 150 mM KCl, 4 mM MgCl2, 2 mM EGTA, 2 mM Na-ATP, 5 mM QX 314, 10 mM HEPES, pH 7.3. With this pipette solution, the Cl− current induced by activation of GABA receptors was recorded as an inward current; QX 314 was used to block Na+ channels. These spontaneous IPSCs could be blocked by the specific GABAA antagonist picrotoxin (200 μM). The pipette was advanced until a seal was obtained over 1 GΩ between the pipette tip and the cell membrane of the identified neuron. The membrane under the pipette tip was then ruptured with a brief suction to obtain a whole-cell patch-clamp configuration and the cell was voltage-clamped at a holding potential of −80 mV. The effects of nociceptin (10, 30, and 100 μM) on spontaneous GABAergic IPSCs were examined at this holding potential; drugs were applied after recording 60 sec of control events; only one concentration of drug was used per neuron (two neurons used per slice). In other experiments, under conditions of synaptic blockade (with 10 μM tetrodotoxin), and 50 μM ±-2-amino-5-phosphonopentanoic acid and 50 μM 6-cyano-7-nitroquinoxaline-2–3(1H,4H)-dione) to prevent glutamatergic postsynaptic currents, exogenous GABA (10 μM) was puffed onto the recording cell [using a patch pipette positioned 10 μm from the cell and a WPI (Sarasota FL) Pneumatic PicoPump] in the absence and presence of nociceptin (100 μM) to determine whether the postsynaptic current was altered. Under these conditions, at least 15 control responses were obtained before drug application; again, each neuron was used only once. Analysis of spontaneous events was performed using MiniAnalysis (version 4.3.1; Synaptosoft, Leonia, NJ) with the minimal acceptable amplitude of events set at 8 to15 pA. The responses to exogenous GABA were analyzed in Clampfit. Results are presented as mean ± S.E.M percentage of control and statistically compared with Student's t test [for significant difference (∗), p < 0.05].
Drugs and Chemicals.
All drugs were purchased from Sigma Chemical (St. Louis MO). Tetrodotoxin was dissolved in acetate buffer, while GABA was prepared in slice perfusate; nociceptin was dissolved in H2O and stored at −20°C until the day of use. Nociceptin was added into the recording chamber by changing the perfusion line to the one containing the drug.
A total of 40 cardiac vagal neurons were recorded using the whole-cell patch-clamp technique. Under recording conditions similar to those used in the experiments, picrotoxin (200 μM) abolished the spontaneous IPSCs, demonstrating these events were due to activation of GABA receptors (control frequency 4.23 ± 0.67 Hz; frequency with picrotoxin 0.45 ± 0.18 Hz, n = 7).
Effects of Nociceptin on Spontaneous IPSCs.
Perfusion with nociceptin caused a dose-dependent decrease in the frequency of the GABAergic IPSCs in cardiac vagal neurons; a significant inhibition was observed with 100 μM nociceptin (Fig.1). At concentrations of 10, 30, and 100 μM, the average frequency of the IPSCs was decreased by 10.03 ± 19.01% (n = 5, p > 0.05), 16.1 ± 15.9% (n = 8, p > 0.05) and 35.6 ± 8.2% (n = 10, p < 0.05), respectively (Fig. 2). This change in frequency is probably due to decreased presynaptic activity, indicating a reduced probability in release of GABA onto cardiac vagal neurons. Perfusion of nociceptin also decreased the amplitude of GABAergic IPSCs in cardiac vagal neurons (see Fig. 1). The inhibition was dose-dependent; at concentrations of 10, 30, and 100 μM, the average amplitude of the IPSCs was decreased by 16.4 ± 7.2% (n = 5, p > 0.05), 41.7 ± 13.8% (n = 8, p < 0.05), and 49.5 ± 5.6% (n = 10, p < 0.05), respectively (Fig. 2). The baseline current did not change upon application of nociceptin (with 100 μM, a 3.1 ± 4.4% inhibition of baseline was observed; with 30 μM, a −0.2 ± 9.1% inhibition was observed). The decrease in IPSC amplitude could reflect either that postsynaptic ORL1 receptors are activated on cardiac vagal neurons decreasing GABAergic responses at concentrations ≥30 μM, or that nociceptin acts presynaptically, leading to changes in neurotransmitter release, or both.
Effect of Nociceptin on Evoked GABA Currents.
To examine whether nociceptin acts at presynaptic or postsynaptic sites to decrease IPSC amplitude, the effects of nociceptin on responses to exogenous applications of GABA were examined. Application of 10 μM GABA onto the cell induced a postsynaptic current averaging 706.8 ± 107.3 pA (constant over 15 applications). The amplitude of this current was reversibly reduced by 36.7 ± 9.8% (n= 6, p < 0.05) in the presence of nociceptin (100 μM) to 467.4 ± 99.5 pA (Fig. 3). These results indicate a novel postsynaptic action of nociceptin on cardiac vagal neurons.
The results from this study demonstrate that nociceptin modulates the activity of cardiac vagal neurons in the nucleus ambiguus by inhibiting their GABAergic input. The effects include a presynaptic mechanism that decreased the frequency (and possibly the amplitude) of GABAergic IPSCs to cardiac parasympathetic neurons and a novel postsynaptic inhibition of GABAergic currents by nociceptin in cardiac vagal neurons.
Heart rate is dominated by the cardioinhibitory parasympathetic nervous system; increases in parasympathetic activity to the heart evoke bradycardia. In vivo studies show that microinjection of bicuculline into the nucleus ambiguus of the brainstem produces a decrease in the heart rate and blood pressure that is mediated by the vagus nerve (DiMicco et al., 1979). This indicates that the nucleus ambiguus is a site of GABA receptor-mediated inhibition of vagal outflow to the heart (DiMicco et al., 1979; Williford et al., 1980). In vitro studies reflect this by demonstrating that cardiac vagal neurons in the nucleus ambiguus are constantly inhibited by spontaneous GABAergic synaptic input; one likely GABAergic pathway may arise from the NTS (Wang et al., 2001a). Evidence from previous studies shows that the nociceptin-evoked bradycardia seen in in vivo experiments (Champion et al., 1997; Giuliani et al., 1997; Champion and Kadowitz, 1997a; Chu et al., 1999a; Kapusta, 2000) may be due in part to activation of parasympathetic outflow to the heart, since this evoked bradycardia is reduced by vagotomy (Giuliani et al., 1997). The results from the experiments here demonstrate the inhibitory effect of nociceptin on activity of parasympathetic preganglionic neurons and suggest that nociceptin may act, in part, to suppress the spontaneous inhibitory GABAergic pathway to cardiac vagal neurons, increasing parasympathetic outflow to the heart and evoking bradycardia.
In this study, the use of a selective ORL1antagonist would have been advantageous to determine the direct effects of nociceptin upon those receptors. However, the few “antagonists” available display disadvantages to their usage. [Phe1ψ(CH2NH)Gly2]-nociceptin(1-13)NH2displays agonist activity centrally at concentrations > 1 μM and is thought to actually be a partial agonist (Emmerson and Miller, 1999; Siniscalchi et al., 1999; Kapusta et al., 1999); the ORL1 receptor antagonist naloxone benzoylhydrazone is nonselective, and a heptadecapeptide known as nocistatin, derived from the same precursor as nociceptin, although shown to block the actions of nociceptin, does not bind to the ORL1 receptor (Schlicker and Morari, 2000). A potent nonpeptidyl-selective ORL1 receptor antagonist (J-113397) has been discussed in another study (Ozaki et al., 2000) but is not yet commercially available.
The mechanisms involved in the nociceptin reduction of presynaptic GABA release are unknown. However, previous work demonstrates that opioid inhibition of GABAergic synaptic currents in the periaqueductal gray is controlled by a voltage-dependent potassium conductance, particularly regarding opioid receptors of the μ-type (Vaughan et al., 1997). Due to the structural and pharmacological resemblance of nociceptin to other endogenous opioids, the effects of nociceptin on voltage-dependent K+ channels in GABAergic neurons that synapse upon cardiac vagal neurons might ascertain the mechanisms underlying nociceptin-mediated inhibition of GABA neurotransmission in cardiac vagal neurons.
The results from this study may also be important therapeutically. In many diseases, such as hypertension and heart failure, cardiac vagal activity is diminished, while restoration of this activity lessens reperfusion-induced arrhythmias (Mendelowitz, 1999). Nociceptin is shown here to increase parasympathetic activity in preganglionic vagal neurons to the heart, suggesting that ORL1receptors may be an effective clinical target in heart disease.
In summary, this study demonstrates that nociceptin decreases the GABAergic neurotransmission to cardiac vagal neurons. This effect includes a presynaptic mechanism acting to decrease the frequency of GABAergic IPSCs and a novel postsynaptic mechanism that decreases the amplitude of spontaneous and evoked GABAergic responses. This may be one mechanism by which nociceptin acts centrally to evoke bradycardia.
This work was supported from National Institutes of Health Grants HL 49965 and 59895 to D.M.
- opioid receptor-like receptor
- nucleus tractus solitarius
- guanosine 5′-O-(3-[35S]thio)triphosphate
- Received July 24, 2001.
- Accepted September 20, 2001.
- The American Society for Pharmacology and Experimental Therapeutics