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CELLULAR AND MOLECULAR

Modulation of Ca²⁺ Channels by Opioid Receptor-Like 1 Receptors Natively Expressed in Rat Stellate Ganglion Neurons Innervating Cardiac Muscle

Department of Anesthesiology (V.R.-V.) and Department of Medicine (A.D.S.), Penn State College of Medicine, Hershey, Pennsylvania; Laboratory of Molecular Physiology (H.L.P.), National Institute on Alcohol Abuse and Alcoholism, Bethesda, Maryland; and Guthrie Vascular Laboratories (B.C.F.), Guthrie Healthcare System, Sayre, Pennsylvania

Received May 9, 2005; accepted June 2, 2005.

	Abstract

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Postganglionic sympathetic nerve terminals innervate cardiac muscle and express opioid receptor-like 1 (ORL1) receptors, the most recently described member of the opioid receptor subclass. ORL1 receptors are stimulated by the endogenous heptadecapeptide nociceptin (Noc). To better understand how the signaling events by Noc regulate sympathetic neuron excitability, the goal of the present study was to determine whether sympathetic stellate ganglion (SG) neurons, innervating the heart, natively express ORL1 opioid receptors and couple to Ca²⁺ channels. SG neurons in adult male rats were retrograde-labeled with a fluorescent tracer via injection of the ventricular muscle employing ultrasound imaging. Thereafter, N-type Ca²⁺ channel modulation was investigated using the whole-cell variant of the patch-clamp technique. Exposure of labeled SG neurons to Noc resulted in a concentration-dependent inhibition of Ca²⁺ currents (with an estimated EC₅₀ of 193 ± 14 nM). Pre-exposure of SG neurons to the ORL1 receptor blocker, [Nphe¹,Arg¹⁴,Lys¹⁵]N/OFQ-NH₂ (UFP-101), significantly decreased the Noc-mediated Ca²⁺ current inhibition. The Ca²⁺ current inhibition was also blocked by pertussis toxin pretreatment, indicating that signaling occurs via G_i/o G proteins. Finally, the full-length ORL1 receptor cDNA in SG neurons was cloned and sequenced. Of the two known alternatively spliced variants in rats, sequencing analysis showed that the ORL1 receptor expressed in SG neurons is the short form. Overall, these results suggest that stimulation of postsynaptic ORL1 receptors by Noc in SG neurons regulate cardiac sympathetic activity.

Although the ORL1 receptor-nociceptin system is known to be involved in pain transmission in the central nervous system (Meunier et al., 2000), recent evidence has shown that opioid receptors are widely distributed in the peripheral nervous system and are capable of eliciting potent cardiovascular responses (for review, see Kapusta, 2000; Malinowska et al., 2002). Noc, for example, evokes a decrease in arterial blood pressure and heart rate when administered intracerebroventricularly, intrathecally, or intravenously (Giuliani et al., 1997, 2000; Salis et al., 2000; Hashiba et al., 2003). Noc has also been found to exert an inhibitory neuromodulation on transmitter release in the heart (Giuliani and Maggi, 1997) and blood vessels (Bucher, 1998; Malinowska et al., 2000). These studies suggest that the presence of ORL1 receptors in the central and peripheral nervous systems regulate cardiac function by modulating autonomic neurotransmission]e.g., inhibition of norepinephrine (NE) release].

Increases in both blood pressure and heart rate, observed following electrical stimulation of preganglionic cardiac efferent sympathetic nerve fibers, were inhibited by Noc in a dose-dependent manner (Malinowska et al., 2001). This response was shown to occur through the stimulation of ORL1 receptors located presynaptically on the postganglionic sympathetic nerve endings. Postganglionic neurons arising from the stellate ganglion (SG) provide the main sympathetic innervation to the heart and play a significant role in regulating cardiac function (Wallis et al., 1996). Accordingly, the purpose of the present study was to determine whether SG neurons, innervating cardiac muscle, possess ORL1 receptors and to examine the signal transduction pathways involved in Ca²⁺ channel modulation. In addition, the identification of the ORL1 receptor splice variant expressed in SG neurons was explored.

	Materials and Methods

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SG Labeling and Isolation. All experimental procedures performed were approved by the Institutional Animal Care and Use Committee (IACUC) at Penn State College of Medicine and conformed to the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. SG neurons were retrograde-labeled employing ultrasound imaging as described previously (Fuller et al., 2004). Briefly, adult male Wistar rats (225–450 g) were anesthetized with intraperitoneal administration of ketamine HCl (75 mg/kg) and xylazine (5 mg/kg). The heart was ultrasonographically imaged using either a Sonoline Sienna or Sequoia C256 Echocardiography system (Siemens Medical Solutions, Mountain View, CA) equipped with a 7.5 or 14 MHz probe, respectively. Thereafter, under echocardiographic guidance, either True Blue (2.5% in H₂O; Molecular Probes, Eugene, OR) or DiIC₁₂(3) (1,1'-didodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate, 1% in DMSO; Molecular Probes) was injected into the ventricular muscle employing a syringe with a 30-gauge needle. The total volume injected ranged from 50 to 100 µl.

Five to 12 days following dye injection, the rats were sacrificed by CO₂ anesthesia and decapitated. The SG was removed and cleared of connective tissue in ice-cold Hanks' balanced salt solution. Thereafter, the SG was incubated for 60 min in a shaking water bath at 35°C in Earle's balanced salt solution with 0.6 mg/ml collagenase Type D (Roche Diagnostics, Indianapolis, IN), 0.4 mg/ml trypsin (Worthington Biochemicals, Freehold, NJ), and 0.1 mg/ml DNase Type I (Sigma-Aldrich, St. Louis, MO). After the incubation period, the cells were dissociated in a culture flask by vigorous shaking, and the dispersed neurons were centrifuged twice for 6 min at 50g and resuspended in minimal essential medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal calf serum, 1% glutamine, and 1% penicillin-streptomycin solution (Invitrogen). Neurons were then plated into 35-mm tissue culture plates coated with poly-L-lysine and stored in a humidified incubator containing 5% CO₂ in air at 37°C.

Fluorescent images of DiI-labeled neurons were captured with an Orca-ER 1394 cooled CCD camera (Hamamatsu Photonics K.K., Hamamatsu City, Japan) and IPLab software (Scanalytics, Fairfax, VA) on an inverted microscope (TE2000; Nikon, Tokyo, Japan) equipped with an epifluorescence unit and G-2E/C filter set (Nikon) containing an excitation filter at 540 ± 12 nm, a dichroic beam splitter of 565 nm (long pass), and an emission filter at 620 ± 30 nm.

Electrophysiology and Data Analysis. For electrophysiological recordings, labeled cells were identified with an inverted microscope (Diaphot 300; Nikon) equipped with an epifluorescence unit and G-2E/C and UV/2A (excitation filter at 355 ± 25 nm, dichroic mirror cut-on at 400-nm long pass, and emission filter at 420-nm long pass; Nikon) filter cube sets for DiI and True Blue, respectively.

Ca²⁺ currents were recorded at room temperature (21–24°C) employing the whole-cell patch-clamp technique. The pipettes were pulled from borosilicate glass capillaries (Corning 7052; Garner Glass, Claremont, CA) on a Flaming-Brown (P-97) micropipette puller (Sutter Instrument Co., San Rafael, CA), coated with Sylgard (Dow Corning, Midland, MI), and fire polished. Whole-cell currents were acquired with a patch-clamp amplifier (Axopatch 1-C; Axon Instruments, Foster City, CA), analog filtered at 5 to 10 kHz (–3 dB; 4-pole Bessel), and digitized using custom designed software (S4) on a Power PC computer (Power Computing Corp., Austin, TX) equipped with a 16-bit analog-to-digital converter board (ITC16; InstruTECH Corporation, Port Washington, NY). Cell membrane capacitance and series resistance (80–85%) were electronically compensated. Data and statistical analyses were performed with the IGOR Pro (Wavemetrics, Lake Oswego, OR) and Prism 4 (GraphPad Software, Inc., San Diego, CA) software packages, respectively. Changes in Ca²⁺ currents were assessed using a standard t test. P < 0.05 was considered statistically significant. Graphs and current traces were produced with the IGOR Pro and Canvas (Deneba Software, Miami, FL) software packages.

The pipette solution contained 120 mM N-methyl-D-glucamine, 20 mM tetraethylammonium hydroxide, 11 mM EGTA, 10 mM HEPES, 10 mM sucrose, 1 mM CaCl₂, 4 mM Mg-ATP, 0.3 mM Na₂ATP, and 14 mM Tris-creatine phosphate. The pH was adjusted to 7.2 with methanesulfonic acid and HCl (20 mM), and the osmolality was 296 to 302 mOsmol/kg. The external solution consisted of 145 mM tetraethylammonium hydroxide, 10 mM HEPES, 15 mM glucose, 10 mM CaCl₂, and 0.0003 mM tetrodotoxin. The pH was adjusted to 7.4 with methanesulfonic acid, and the osmolality was 317 to 323 mOsmol/kg.

The concentration-response curve for Noc was determined by sequential application of increasing concentrations of the agonist. To avoid effects of desensitization by Noc, no more than three concentrations were tested in each cell, and electrophysiological recordings from neighboring cells were avoided. In most cases, prior to the end of the recordings, 3 µM Noc (e.g., third application) was applied to obtain the maximal Ca²⁺ current inhibition. In this set of experiments, a sequential application of Noc was not performed until the Ca²⁺ current amplitude returned to the control value. The results were pooled, and each point represents the mean ± S.E.M. The concentration-response curve was fit to the Hill equation: I = 1/[1 + (k/[Noc])ⁿ] where I is the normalized Ca²⁺ current inhibition, k is a constant, [Noc] is Noc concentration, and n is Hill factor.

Solutions and Drugs. Stock solutions of Noc, [D-Ala²]-deltorphin II, U-50488, and endomorphin-1 (all from Tocris Cookson, Ellisville, MO) and vasoactive intestinal peptide (VIP; Phoenix Pharmaceuticals, Belmont, CA) were prepared as stock solutions in H₂O and diluted to their final concentration just prior to use. PTX (List Biological Laboratories, Inc., Campbell, CA) was prepared in H₂O and added to the culture medium (12–20 h) at a final concentration of 500 ng/ml. UFP-101, a kind gift from Dr. Giro Calo (University of Ferrara, Ferrara, Italy), was also prepared in H₂O and applied via the perfusion system at least 10 min before seal formation and maintained for the duration of the experiment. Drug application to the cells under study was performed by placing a custom-designed gravity-fed perfusion system approximately 100 µm from the neuron.

Isolation of the Full-Length Rat ORL1 Receptor cDNA. Total RNA was isolated from SG obtained from adult Wistar rats, described above. Thereafter, total RNA was purified with the RNeasy miniprep kit (QIAGEN, Valencia, CA). Approximately 1 µg of total RNA was used to generate oligo(dT) primed first-strand cDNA using the Advantage RT for polymerase chain reaction (PCR) kit (BD Biosciences Clontech, Palo Alto, CA). Primers for the full-length PCR were generated based on the ORL1 receptor sequence from GenBank account no. NM_031569 [GenBank] . The primers used were: 5'-GAT CGG ATC CAC CAT GGA GTC CCT CTT TCC TGC TC and 3'-GAT CCT CGA GTC ATG CTG GCC GTG GTA CTG TC. The restriction enzyme sites for BamHI and XhoI were added to the N- and C-terminal primers, respectively. The full-length ORL1 receptor PCR product was generated using Pfu Turbo DNA polymerase (Stratagene, La Jolla, CA) and subcloned with the Zero Blunt TOPO PCR Cloning Kit (Invitrogen). Positive clones were sequenced employing BigDye terminators (Applied Biosystems, Foster City, CA) and ABI 377 DNA sequencer (Applied Biosystems). The nucleotide sequence was deposited in GenBank (AY152731 [GenBank] ).

View larger version (42K): [in this window] [in a new window]   Fig. 1. Phase contrast (A) and fluorescence (B) image of SG neurons isolated from an adult rat 6 days following DiI injection to the cardiac muscle. The fluorescence image was obtained with a filter set specific for DiI (540-nm excitation and 620-nm emission). The fluorescence image was pseudocolored; scale bar represents 20 µm.

	Results

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View larger version (16K): [in this window] [in a new window]   Fig. 2. Effect of opioid receptor agonists on Ca2+ channel currents and facilitation of nociceptin-mediated Ca2+ current inhibition. A, time course of Ca2+ current amplitudes of labeled SG neurons were recorded every 10 s by applying a 50-ms depolarizing test pulse to +10 mV from a holding potential of –80 mV. Filled bars indicate application of 10 µM endomorphin-1, 3 µM nociceptin, and 10 µM U-50488. B, superimposed current traces from SG neuron in A recorded before and during opioid receptor agonist application. C, summary of mean (±S.E.M.) Ca2+ current inhibition produced by each opioid receptor agonist: nociceptin (ORL1), Deltorphin II (, 2 µM), U-50488 (), and Endomorphin-1 (µ). Numbers in parentheses indicate the number of experiments.

The next set of experiments was undertaken to determine the concentration-response relationship for Noc-stimulated ORL1 Ca²⁺ current inhibition in labeled SG neurons. The Ca²⁺ currents were recorded as described for Fig. 2A. Figure 3, A and B, illustrates that exposure of 0.03 and 1 µM Noc caused a 7 and 75% inhibition of Ca²⁺ currents, respectively. The Noc concentration-response curve is shown in Fig. 3C. Results are expressed as Ca²⁺ current inhibition (normalized to 3 µM Noc) plotted against log[nociceptin] and then fitted using the Hill equation. The estimated EC₅₀ was 193 ± 14 nM (n = 3–13 neurons).

View larger version (19K): [in this window] [in a new window]   Fig. 3. Nociceptin concentration-response relationship of labeled SG neurons and sensitivity to UFP-101. A, time course of Ca2+ current amplitude inhibition acquired from the sequential application of 0.03 and 1 µM nociceptin in labeled SG neurons. Currents were evoked every 10 s by a single 50-ms test pulse to +10 mV from a holding potential of –80 mV. B, superimposed current traces from the neuron in A represent those before and during agonist application. C, nociceptin-mediated Ca2+ current inhibition. The peak Ca2+ current inhibition was normalized to the peak current evoked by 3 µM nociceptin. Each data point represents the mean (±S.E.M.). D, summary graph showing the mean (±S.E.M.) Ca2+ current inhibition produced by 1 µM nociceptin and 1 µM nociceptin + 10 µM UFP-101. Inset shows superimposed current traces before (control) and after application of nociceptin + UFP-101. Numbers in parentheses indicate the number of experiments.

In this set of experiments, the signaling components involved in ORL1 receptor-mediated Ca²⁺ channel inhibition were examined. ORL1 receptors typically couple to the PTX-sensitive G_i/o G protein subfamily. Figure 4A shows the time course of peak Ca²⁺ currents following application of Noc (3 µM) and VIP (10 µM). VIP stimulates a G protein-coupled receptor subtype that preferentially couples to the PTX-insensitive G_s G protein subfamily (Zhu and Ikeda, 1994). The inset in Fig. 4A shows the Ca²⁺ current traces before and after agonist exposure. The Ca²⁺ current inhibition mediated by Noc and VIP were 64 ± 4% (S.E.M., n = 10) and 54 ± 5% (S.E.M., n = 6), respectively. A separate group of SG neurons was pretreated overnight with PTX, and the time course of peak Ca²⁺ currents is shown Fig. 4B. The plot and inset show that application of Noc resulted in a small inhibition of Ca²⁺ currents when compared with control neurons. Conversely, when the neuron was exposed to VIP, Ca²⁺ current inhibition was similar to that observed in control neurons. Figure 4C is a summary that illustrates PTX pretreatment disrupted the coupling between ORL1 receptors and Ca²⁺ channels such that the Noc-mediated Ca²⁺ current inhibition was significantly decreased (P < 0.05).

View larger version (25K): [in this window] [in a new window]   Fig. 4. Nociceptin-mediated Ca2+ current inhibition is PTX-sensitive and voltage-dependent. A and B, time course of Ca2+ current amplitudes of labeled SG neurons acquired as in Fig. 2 in control (A) and PTX-treated (500 ng/ml, B) labeled SG neurons. Filled bars indicate application of 3 µM nociceptin and 10 µM VIP. C, summary of mean (±S.E.M.) Ca2+ current inhibition produced by nociceptin and VIP. Numbers in parentheses indicate the number of experiments. *, P < 0.05 compared with control, Student's t test. D, superimposed Ca2+ current traces evoked with the double-pulse voltage protocol (shown at top) in the absence (bottom traces) and presence (top traces) of 3 µM nociceptin in a labeled SG neuron.

Next, we wanted to determine whether the Noc-mediated inhibitory effects on Ca²⁺ currents occurred in a voltage-dependent (VD) manner. N-type Ca²⁺ channel currents are modulated by various neurotransmitters resulting in "kinetic slowing" of the rising Ca²⁺ currents during a depolarizing test pulse (for review, see Ikeda and Dunlap, 1999). The kinetic slowing is thought to be the result of a VD relief of channel block during the test pulse. The VD relief of Ca²⁺ channel inhibition leads to "facilitation" of Ca²⁺ currents and can be observed by applying a "double-pulse" voltage protocol (Ikeda, 1991). Employing this voltage paradigm, Ca²⁺ current amplitude is measured from the test pulses (+10 mV) occurring after and before the conditioning pulse to +80 mV. One hallmark of VD inhibition is an enhanced postpulse current amplitude. Figure 4D shows superimposed Ca²⁺ current traces evoked with the double-pulse voltage protocol (shown at the top) before (top trace) and after (bottom trace) Noc (3 µM) exposure. In the absence of agonist, it can be seen that the conditioning pulse had a minor effect on the post-pulse current amplitude. The presence of a "tonic facilitation" in sympathetic neurons has been shown to result from a small degree of basal G protein activation (Ikeda, 1991; Ruiz-Velasco and Ikeda, 2000). Following bath application of Noc, the Ca²⁺ currents displayed both a VD block and kinetic slowing as evidenced by the biphasic rising phase (top trace). It should be mentioned that the conditioning pulse did not fully restore current amplitude in the presence of Noc. This is possibly a result of a voltage-independent modulation of Ca²⁺ channels and involves second messenger pathways (Ikeda and Dunlap, 1999; Beedle et al., 2004).

At least two ORL1 receptor splice variants have been reported to be expressed in rat sympathetic (superior cervical and lumbar) and sensory neurons (Xie et al., 1999). The long variant contains an 84-base pair insertion in the receptor's second extracellular loop domain. This additional sequence contains a putative N-linked glycosylation site. In this set of experiments, we wanted to ascertain which ORL1 receptor subtype was present in SG neurons. Following mRNA isolation and PCR amplification with primers (see Materials and Methods), the receptor was cloned. Figure 5 shows the nucleotide and deduced amino acid sequence of the cloned ORL1 receptor in SG neurons. Of the eight clones obtained, none contained the 84-base insertion described above. The boxed amino acid sequences represent putative transmembrane segments. This sequence corresponds to the short splice variant of the ORL1 receptor (Xie et al., 1999). When compared with other deduced amino acid sequences derived from short splice variant forms isolated from rat brain (GenBank account nos. NM_031569 [GenBank] , D16438 [GenBank] , and U07871 [GenBank] ), there is a 100% homology. Thus, from these results, the long splice variant does not appear to be expressed in rat SG neurons.

View larger version (39K): [in this window] [in a new window]   Fig. 5. Rat SG ORL1 receptor cDNA nucleotide sequence and its predicted amino acid sequence. The nucleotide sequence is numbered from the start codon ATG, and the stop codon is represented by the asterisk. The receptor's putative seven transmembrane domains are boxed.

	Discussion

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The series of aforementioned experiments explored the coupling mechanisms between the opioid ORL1 receptor and Ca²⁺ channels in rat SG neurons. Administration of the endogenous ORL1 receptor ligand, Noc, has been reported to exert vasopressor effects. The cardiovascular actions produced by Noc are a result of the peptide's ability to modulate autonomic neurotransmission at several levels (Kapusta, 2000). For instance, Noc can inhibit NE release in sympathetic neurons innervating blood vessels (Malinowska et al., 2000), vas deferens (Bigoni et al., 1999), and rat tail artery (Bucher, 1998). In addition, the hypotensive and bradycardiac effects of Noc administration have been shown to correlate with decreases in plasma NE levels (Hashiba et al., 2003). Finally, a recent report found that ORL1 receptors are present in rat sympathetic neurons innervating cardiac muscle (Malinowska et al., 2001). Since ORL1 receptors and N-type Ca²⁺ channels have been found to form a physical complex (Beedle et al., 2004), it was hypothesized that Noc-stimulated ORL1 receptors would modulate Ca²⁺ channels in SG neurons. Thus, we took advantage of our ability to retrograde label SG neurons with a fluorescent tracer in a noninvasive manner to focus on the signaling mechanisms underlying the modulation of Ca²⁺ channels by Noc-stimulated ORL1 receptors.

Our initial findings suggest that in acutely isolated SG neurons the three classic opioid receptors (µ, , and ) are not expressed in the soma. These results are supported by previous studies that reported opioid peptides modulate preganglionic rather than postganglionic nerve activity in rat (Mo et al., 1994), cat (Prosdocimi et al., 1986), and guinea pig (Ledda et al., 1984) SG neurons. Although these earlier studies did not focus on ion channel modulation, they provide evidence that suggests these opioid receptor subtypes do not modulate postganglionic transmission in SG neurons. An alternative explanation may be that stimulation of these receptors does not lead to N-type Ca²⁺ channel modulation. Furthermore, studies need to be performed to explore this possibility.

The measured potency of the Noc-mediated Ca²⁺ current inhibition in this study was comparable with the values reported by others in pyramidal neurons (Knoflach et al., 1996) and cultured cells (Morikawa et al., 1998). However, the EC₅₀ value was greater than that found in periaqueductal gray neurons (Connor and Christie, 1998), rat dorsal root ganglion (DRG) neurons (Beedle et al., 2004), and nearly 200-fold higher in rat superior cervical ganglion (SCG) neurons (Larsson et al., 2000). First, one possible reason for the discrepant findings between sympathetic SCG and SG neurons may be the rat strain employed. Wistar rats were used in the present study, whereas Sprague-Dawley rats were employed by Larsson et al. (2000). Second, we recorded Ca²⁺ currents from neurons that were incubated overnight following cell dissociation, whereas Larsson and colleagues measured Ca²⁺ channel currents within 3 to 8 h of cell isolation. And third, differences in ORL1 receptor expression density between both neuron cell types may also account for these discrepancies. Finally, the difference in potency between the present study and that in rat DRG neurons (Beedle et al., 2004) may be a result of the ORL1 receptor splice variant expressed in each neuron type. For example, a recent molecular study (discussed below) found that ORL1 mRNA is present in both SCG and DRG (Xie et al., 1999). However, expression of the long splice variant is dominant in the former, whereas expression of the shorter splice variant is greater in the latter.

In the present study, the signaling elements involved in coupling ORL1 receptors and Ca²⁺ channels were also investigated. Because ORL1 receptors and opioid receptors, in general, are well established to couple to PTX-sensitive G_i/o protein subunits (New and Wong, 2002), we found that Ca²⁺ channel inhibition was significantly decreased in SG neurons pretreated with the toxin. Similar findings have been previously published (Morikawa et al., 1998; Connor et al., 1999; Larsson et al., 2000; Beedle et al., 2004; Yeon et al., 2004). Nevertheless, it has been reported that ORL1 receptors are also capable of coupling to other PTX-insensitive G protein subunits to modulate other effectors (Chan et al., 1998; Chan and Wong, 2000).

In this study, the modulation of N-type Ca²⁺ channel currents by Noc-stimulated receptors was further examined. The VD inhibition of Ca²⁺ currents is membrane-delimited and mediated by the free G dimers that are released following receptor stimulation (Herlitze et al., 1996; Ikeda, 1996). Exposure of labeled neurons to Noc resulted in the VD inhibition of Ca²⁺ currents, as observed by kinetic slowing and an increase in postpulse facilitation. Although it is not exactly clear how binding of G is coupled to the activation machinery of N-type Ca²⁺ channels, our observations also point to G as the active moiety that modulates N-type Ca²⁺ channels. These observations have been reported in other neurons and expression systems (Morikawa et al., 1998; Connor et al., 1999; Larsson et al., 2000; Beedle et al., 2004; Yeon et al., 2004). In a more detailed study of Noc's modulation of Ca²⁺ channels, evidence has been presented to indicate that ORL1 receptors form a physical signaling complex with N-type Ca²⁺ channels that results in tonic VD inhibition (Beedle et al., 2004). In that report, it was shown that the agonist-independent, tonic VD inhibition of N-type Ca²⁺ channels occurred in both rat DRG neurons and tsa-201-transfected cells. The tonic inhibition was more noticeable in small DRG than in large DRG neurons (Beedle et al., 2004).

Pharmacological studies employing ORL1- opioid receptor chimeras have shown that the second extracellular loop plays an essential role in ORL1 receptor binding and activation (Mollereau et al., 1999; New and Wong, 2002). Previous reports have shown that at least two ORL1 opioid receptor splice variants (long and short) are present in rats (Wang et al., 1994; Xie et al., 1999). The long splice variant contains an additional 84-base pair sequence (28 amino acids) that codes for an N-linked glycosylation sequence in the second extracellular loop. Currently, expression or functional data exploring the consequence of this additional sequence is not available and needs to be further examined. Interestingly, Xie and colleagues (1999) found that the ratio of long to short splice variant expression, as measured by mRNA levels, in rat sympathetic lumbar and SCG neurons was approximately 5:1. More recent studies indicate the existence of several additional splice variants (Curro et al., 2001) and an 81-base insertion in the second extracellular loop due to retention of intron 3 (Xie et al., 2000). This retained intron is in the same location as the 84-base insertion mentioned above but leads to an inframe stop resulting in a truncated receptor protein. However, in the present study, only the short splice variant was detected in mRNA samples isolated from SG neurons. Thus, it appears that ORL1 receptor expression in rat peripheral sympathetic neurons may occur in a differential manner and may also help explain the differences in N-type Ca²⁺ channel modulation between SG and SCG (Larsson et al., 2000) or DRG neurons (Beedle et al., 2004) indicated above.

Recent observations by Malinowska and colleagues (Malinowska et al., 2000, 2001, 2002) have shown that intravenous exposure of Noc in anesthetized rats led to a decrease in blood pressure and heart rate. As ORL1 receptors are expressed in central and peripheral nervous systems, the Noc-induced effects likely occur at several levels. For instance, Noc has been shown to inhibit the inotropic response produced by electrically evoked NE release from sympathetic nerve endings in the guinea pig isolated atrium (Giuliani and Maggi, 1997). Thus, the findings of the present study further aid in elucidating some of the underlying mechanisms that are involved in the regulation of the cardiovascular system following Noc administration. This study focused on SG neurons, the sympathetic input to cardiac muscle. These findings suggest that the Noc-induced hypotension and bradycardia, observed by others (Giuliani et al., 1997; Malinowska et al., 2001), is the result of neurotransmitter release modulation via N-type Ca²⁺ channel inhibition in SG neurons. Therefore, the presence of ORL1 receptors in SG neurons likely regulate Noc-induced cardiac inotropism.

In summary, our results show that sympathetic SG neurons innervating cardiac muscle express ORL1 receptors that modulate N-type Ca²⁺ channels following exposure to Noc. The ORL1 receptor-Ca²⁺ channel coupling mechanism is PTX-sensitive and blocked by the UFP-101 compound. Moreover, the inhibition of N-type Ca²⁺ channels is VD and membrane-delimited. Cloning experiments of the ORL1 receptor revealed one receptor subtype or one splice variant in adult rats.

	Acknowledgements

 
We thank Dr. Giro Calo (Department of Experimental and Clinical Medicine, Section of Pharmacology, University of Ferrara, Ferrara, Italy) for kindly supplying UFP-101.

	Footnotes

 
This study was supported by National Institutes of Health Grant HL-074311 to V.R.-V.

doi:10.1124/jpet.105.089284.

ABBREVIATIONS: ORL1, opioid receptor-like 1; Noc, nociceptin; PTX, pertussis toxin; NE, norepinephrine; SG, stellate ganglion; DiIC₁₂(3), 1,1'-didodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate; U-50488, trans-(–)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide hydrochloride; VIP, vasoactive intestinal peptide; UFP-101, [Nphe¹,Arg¹⁴,Lys¹⁵]N/OFQ-NH₂; PCR, polymerase chain reaction; VD, voltage-dependent; DRG, dorsal root ganglion; SCG, superior cervical ganglion.

Address correspondence to: Dr. Victor Ruiz-Velasco, Department of Anesthesiology, H187, Penn State College of Medicine, Hershey, PA 17033-0850. E-mail: vruizvelasco{at}psu.edu

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