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Local opiate receptors in the sinoatrial node moderate vagal bradycardia
Autonomic Neuroscience: Basic and Clinical 87 (2001) 9–15 www.elsevier.com / locate / autneu
Local opiate receptors in the sinoatrial node moderate vagal bradycardia a b b b, Martin Farias , Keith Jackson , Amber Stanfill , James L. Caffrey * a
b
Department of Biology, University of Texas /Brownsville, Brownsville, TX 78520, USA Department of Integrative Physiology, Cardiovascular Research Institute, University of North Texas Health Science Center, UNTHSC-FW, 3500 Camp Bowie Boulevard, Fort Worth, TX 76106, USA Received 26 June 2000; received in revised form 6 September 2000; accepted 8 September 2000
Abstract Met-enkephalin-arg-phe (MEAP) interrupts vagal bradycardia when infused into the systemic circulation. This study was designed to locate the opiate receptors functionally responsible for this inhibition. Previous observations suggested that the receptors were most likely located in either intracardiac parasympathetic ganglia or the pre-junctional nerve terminals innervating the sinoatrial node. In this study 10 dogs were instrumented with a microdialysis probe inserted into the sinoatrial node. The functional position of the probe was tested by briefly introducing norepinephrine into the probe producing an increase in heart rate of more than 30 beats / min. Vagal stimulations were conducted at 0.5, 1,2 and 4 Hz during vehicle infusion (saline ascorbate). Cardiovascular responses during vagal stimulation were recorded on-line. MEAP was infused directly into the sinoatrial node via the microdialysis probe. The evaluation of vagal bradycardia was repeated during the nodal application of MEAP, diprenorphine (opiate antagonist), and diprenorphine co-infused with MEAP. MEAP introduced into the sinoatrial node via the microdialysis probe reduced vagal bradycardia by more than half. Simultaneous local nodal blockade of these receptors with the opiate antagonist, diprenorphine, eliminated the effect of MEAP demonstrating the participation by opiate receptors. Systemic infusions of MEAP produced a reduction in vagal bradycardia nearly identical to that observed during nodal administration. When local nodal opiate receptors were blocked with diprenorphine, the systemic effect of MEAP was eliminated. These data lead us to suggest that the opiate receptors responsible for the inhibition of vagal bradycardia are located within the sinoatrial node with few, if any, participating extra-nodal or ganglionic receptors. 2001 Elsevier Science B.V. All rights reserved. Keywords: Enkephalins; Sinoatrial node; Bradycardia; Vagus nerve; Microdialysis
1. Introduction Although the myocardium contains a variety of opioids, their function in heart is not well understood. Howells et al. (1986) originally found more proenkephalin mRNA in rat heart than in comparable samples of rat brain or adrenal gland. Despite rather limited concentrations of the fully processed met- and leu-enkephalin in myocardial extracts, the abundant message strongly suggested that the heart produced enkephalin. This hypothesis was subsequently confirmed by several laboratories who demonstrated that the RNA was translated and the product secreted in vitro (Springhorn and Claycomb, 1989; Low et al., 1990; Springhorn and Claycomb, 1992). In addition, large concentrations of immunoreactivity corresponding to intact proenkephalin and its C-terminal fragments, Peptide-B and *Corresponding author. Tel.: 11-817-735-2085; fax: 11-817-7355084. E-mail address: [email protected] (J.L. Caffrey).
met-enkephalin-arg-phe (MEAP), were identified in the dog heart (Barron et al., 1992; Mateo et al., 1995). Weitzell et al. (1984) initially demonstrated that enkephalins inhibited vagal transmission in isolated hearts. They reported that morphine, met-enkephalin, and D-Ala 2 , 5 D-Leu -enkephalin all reduced vagal bradycardia in rabbit hearts and the effect of each was reversed by the opiate antagonist, naloxone (Weitzell et al., 1984). Enkephalins have also been reported to suppress vagally induced bradycardia in vivo and may therefore function as governors of vagal control (Musha et al., 1989; Caffrey et al., 1995; Pokrovsky and Oadchiy, 1995; Caffrey, 1999). During adrenergic receptor blockade, a reduction in vagal bradycardia was observed when cardiac sympathetic nerves were activated. This vagolytic effect was reversed in part by the opiate antagonist naloxone. Since enkephalin produced a similar vagal inhibition, the authors suggested the impaired vagal function resulted from endogenous opiates (Koyanagawa et al., 1989). Our laboratory reported that MEAP was more effective than met-enkephalin in
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M. Farias et al. / Autonomic Neuroscience: Basic and Clinical 87 (2001) 9 – 15
dogs and effectively attenuated vagally induced bradycardia when infused systemically at picomolar rates (Caffrey et al., 1995). Diprenorphine inhibited the effect of MEAP and provided support for participation by opiate receptors. We also have been able to demonstrate that MEAP can inhibit vagal control of heart rate, contractile force and coronary blood flow (Caffrey, 1999). The fact that enkephalins can inhibit vagal bradycardia appears well supported by the prior studies. The location of the opioid receptor that mediates this effect, however, remains unclear. MEAP did not alter the negative chronotropic effect of the direct acting, muscarinic agonist, methacholine (Caffrey et al., 1995). The inability to alter the response to methacholine suggested that MEAP exerted its effect at a site in the efferent vagal tract that is proximal to the nodal, muscarinic receptors. This narrows the location of these opiate receptors to sites within the intracardiac parasympathetic ganglia or at prejunctional sites (e.g. vagal nerve terminals) within the sinoatrial node (Caffrey et al., 1995). Since the two primary candidates, the sinoatrial node and the intracardiac parasympathetic ganglia are anatomically adjacent to one another and the sinoatrial node artery perfuses both, unambiguously assigning target status to one or both has been technically difficult. The following study makes an innovative use of microdialysis to more precisely locate the opioid receptors responsible for the ability of MEAP to modify vagal bradycardia.
2. Materials and methods Ten dogs were assigned to various experimental protocols. All protocols were approved by the Institutional Animal Care and Use Committee and were in compliance with the NIH Guide for the care and use of laboratory animals. The dogs were anesthetized with pentobarbital sodium (32.5 mg / kg). The dogs were intubated and mechanically ventilated initially at 225 ml / min per kg with room air. Fluid filled catheters were then inserted into the right femoral artery and vein and advanced respectively into the descending aorta and inferior vena cava. The arterial line was attached to a Statham PD23XL pressure transducer and heart rates were measured continuously on-line (MacLabs). The venous line was positioned above the diaphragm and was used to administer supplemental anesthetic and to infuse peptides systemically. The acid– base balance and the blood gases were determined regularly with an Instrumentation Laboratories Blood Gas Analyzer. The pO 2 (90–120 mmHg), the pH (7.35–7.45) and the pCO 2 (30–40 mmHg) were adjusted to normal by administering supplemental oxygen, bicarbonate or modifying the minute volume. Supplemental anesthetic was administered intravenously as required. The right and left vagus nerves were isolated through a midline surgical incision and ligated tightly with umbilical
tape for later retrieval. Surgical anesthesia was carefully monitored, and a single dose of succinylcholine (50 mg / kg) was administered intravenously to temporarily reduce involuntary movements of the muscles during the 10–15 min required for electrosurgical incision of the chest and removal of the right ribs two to five. The heart was exposed from the right aspect. The pericardium was opened and the dorsal pericardial margins were sutured to the body wall to provide a pericardial cradle. A 25 g stainless steel spinal needle containing a microdialysis line was inserted into the center of the long axis of the sinoatrial node. The needle was removed and the probe was then positioned so that the dialysis window was within the substance of the sinoatrial node. The microdialysis probes were constructed of a single 1 cm length of dialysis fiber derived from a Clirans 10 (Terumo Medical Corporation, Sumerset, NJ) artificial kidney. The dialysis tubing had a 300 mm inner diameter (ID), a 305 mm outer diameter (OD) and a molecular weight cutoff of 5000. The inflow and outflow lines were composed of hollow silica tubing (SGE, Austin, TX) glued into the dialysis fiber (Van Wylen et al., 1990). Norepinephrine (1 nmol / ml) was diluted in saline and 0.1% ascorbic acid (vehicle) and perfused into the microdialysis probe at 4 ml / min in order to confirm functionally the position of the probe in the sinoatrial node. If properly positioned, the heart rate routinely increased by 30 beats / min (bpm) or more within 30 s. This tachycardia was not observed when norepinephrine was introduced into probes placed elsewhere in the atrial wall. The norepinephrine was flushed out and the probe was perfused with vehicle for 60 min. Vehicle or peptide was then perfused through the probe while the right vagus nerve was stimulated at 0.5 Hz for 15 s at a supra-maximal voltage (usually 15 V). The system was allowed 1 min and 45 s to recover. A complete frequency response curve was then constructed by repeating the 2-min stimulus / recovery protocol sequentially at 1, 2, and 4 Hz. Fifteen minutes were subsequently allowed to elapse between each series of frequency responses. When employed, systemic peptide infusions were administered intravenously at 3 nmol / min per kg. This infusion rate was previously determined to provide the maximal inhibition of the vagal control of heart rate. All peptides were prepared in saline with 0.1% ascorbic acid to prevent oxidation and were infused for 5 min prior to beginning any stimulation protocols.
2.1. Protocol I: Intranodal MEAP and intranodal diprenorphine This initial protocol consisted of four treatments designed to determine if local opiate receptors in the sinoatrial node were responsible for the MEAP mediated inhibition of vagal bradycardia. The experiment was also designed to verify that opiate receptors were in fact responsible for this effect. All treatments were infused at
M. Farias et al. / Autonomic Neuroscience: Basic and Clinical 87 (2001) 9 – 15
4ul / min into the substance of the sinoatrial node using a micro-syringe pump. Saline ascorbate served as vehicle control for comparison with MEAP (1 nmol / ml), the opiate antagonist diprenorphine (0.22 nmol / ml) and the co-infusion of both agonist and antagonist together. MEAP was selected for these studies because it is found in high concentrations in the dog heart, it is a natural agonist, and it is more effective than methionine enkephalin (Barron et al., 1992; Caffrey et al., 1995; Mateo et al., 1995). Since we were unsure of which opiate receptor might be responsible for the expected effects, diprenorphine was chosen as the opiate antagonist in these studies because of its high affinity and relative non-selectivity at each of the three opiate receptor subtypes (Sadee et al., 1982).
2.2. Protocol II: Systemic MEAP and intranodal diprenorphine This protocol was designed to determine if the intracardiac parasympathetic ganglia also served as effective targets where MEAP could modify vagal bradycardia. The four treatments from Protocol I were repeated to verify that the nodal diprenorphine applied was adequate to block nodal opiate receptors. MEAP (3 nmol / min per kg) was then infused systemically to compare its effect with nodal administration. Prior studies have narrowed down the likely location of the opiate receptors responsible for the inhibition of bradycardia to the sinoatrial node and / or the intracardiac parasympathetic ganglia. As a result, MEAP was also administered intravenously while local nodal opiate receptors were blocked with the opiate receptor antagonist diprenorphine (0.22 nmol / ml) delivered via the microdialysis probe.
2.3. Materials Methionine enkephalin-arg-phe was synthesized by American Peptide Co, Sunnyvale, CA and diprenorphine was obtained from the National Institutes for Drug Abuse. All microdialysis probes were fabricated by David G.L. Van Wylen of St. Olaf College in St. Olaf Minnesota.
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2.4. Statistics The data are expressed throughout as means and standard errors. Differences were evaluated with an analysis of variance and multiple post-hoc comparisons were made with the aid of Tukey’s protected-t (GB-STATE, Dynamic Microsystems, Silver Spring, MD). Repeated measures designs were used where appropriate. P,0.05 was accepted as statistically different.
3. Results Table 1 provides the resting cardiovascular parameters for all animals across all treatments. As indicated there were no changes in baseline function prior to stimulation. Fig. 1 provides a schematic representation of the spatial relationships among the ganglion cells, the sinoatrial (SA) node, and their common vascular supply, the SA node artery. The SA node in the average 20 kg dog appears to be approximately 10–20 mm long and 2–3 mm in diameter. The structures are not drawn to scale and the fat pad is much larger than the node. In order to verify the functional position of the nodal microdialysis probe, norepinephrine (100 ng / ml) was infused briefly at 4 ml / min. A rapid increase in heart rate between 30 and 60 beats was routinely observed and was considered sufficient evidence of optimal positioning of the probe. If norepinephrine was introduced into probes placed elsewhere in the atrial free wall no comparable increase in heart rate was observed Fig. 2 illustrates the change in heart rate in 10 dogs during vagal stimulations at 0.5, 1, 2, and 4 Hz under the four different treatments of Protocol I. Infusion of saline ascorbate into the nodal probe had no effect on the resting heart rate. Stimulation of the right vagus nerve produced average declines in heart rate of 13, 21, 35 and 67 bpm, respectively. After 15 min, MEAP was introduced into the probe and perfused for an additional 5 min prior to stimulation. MEAP had no effect on the resting heart rate. The subsequent vagally induced bradycardia however, was reduced by more than one-half of that observed under
Table 1 Resting cardovascular indices
Heart rate (bpm) MAP (mmHg) Systolic (mmHg) Diastolic (mmHg)
Control
Nodal MEAP
13867
13067
11666
Nodal diprenorphine
Nodal MEAP nodal diprenorphine
Systemic MEAP
Systemic MEAP nodal diprenorphine
12869
13467
134610
143610
11466
11467
10966
10968
11468
14468
14268
140610
13668
133611
138611
10465
10165
10267
9765
9768
10168
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M. Farias et al. / Autonomic Neuroscience: Basic and Clinical 87 (2001) 9 – 15
Fig. 1. Schematic representation of the spatial relationships in the area of the SA node. The structures are not drawn to scale.
control conditions. With MEAP in the dialysis probe, stimulation of the vagus nerve produced graded declines in heart rate of 6, 10, 17, and 28 bpm. After flushing the probe with saline, the vagal response routinely returned to normal within 5 min. Fifteen minutes after flushing out the
Fig. 2. Changes in heart rate mediated by right vagal stimulation under control conditions during vehicle applied by microdialysis (d), during nodal MEAP applied by microdialysis (j), after nodal opiate receptor blockade with diprenorphine applied by microdialysis probe (m), and MEAP1diprenorphine applied by microdialysis probe (.). Values are means; error bars have been omitted for clarity; n58–10. * Significantly different from the three lower curves at the same frequency.
peptide, the opiate antagonist, diprenorphine was introduced into the probe. Diprenorphine had no effect on the resting heart rate and the heart rate response during vagal stimulation was not statistically different from that observed under control conditions. When MEAP and diprenorphine were co-infused into the sinoatrial node, there was again no effect on the resting heart rate. Stimulation of the right vagus nerve during the combined treatment produced responses that were not statistically different from the control stimulations. Since local diprenorphine blocked the effect of local MEAP, the effect of MEAP must be mediated by nodal opiate receptors. The reduced vagal bradycardia produced by MEAP applied by microdialysis to the node was statistically different from the other three treatments in this protocol. Fig. 3 illustrates the change in heart rate for six dogs with two additional treatments. The first four treatments are the same as in protocol one. The additional treatments included systemic MEAP (3 nmol / min per kg, iv) and systemic MEAP combined with local diprenorphine administered by dialysis into at the sinoatrial node. These two treatments had no effect on resting heart. The results from the first four treatments were the same as those in Fig. 1, but were repeated to provide a direct comparison to the earlier experiments and to verify that the dose of diprenorphine was sufficient to block the nodal effect of MEAP. The dose of MEAP used systemically was previously determined to produce the maximal inhibition of vagal bradycardia (Caffrey et al., 1995). Systemic MEAP infusions reduced vagal bradycardia to an extent similar to that observed after the nodal application of the peptide. However, when diprenorphine was reintroduced into the nodal probe the vagolytic effect of systemic MEAP was
M. Farias et al. / Autonomic Neuroscience: Basic and Clinical 87 (2001) 9 – 15
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Fig. 3. Changes in heart rate mediated by right vagal stimulation under control conditions (d), during intravenous MEAP infusion (j), and during intravenous MEAP combined with nodal opiate receptor blockade produced by diprenorphine applied by microdialysis (m). Values are means; error bars have been omitted for clarity; n56–10. * Significantly different from the two lower curves at the same frequency.
completely eliminated. During this nodal opiate receptor blockade, vagal stimulations produced graded declines in heart rate indistinguishable from control. Although unlikely, we considered the possibility that nodal diprenorphine might also reach the nearby parasympathetic ganglion cells by retrograde diffusion along nerve tracks between the ganglion and the SA node. To test this hypothesis, the systemic effect of MEAP was evaluated in two dogs before and after infiltrating the ganglionic fat pad with diprenorphine. The dose of diprenorphine was equal to the entire content perfused through the probe during each of the protocol II experiments described above. Under these circumstances, the vagolytic effect of systemic MEAP was identical before and after the ganglionic administration of diprenorphine. Unlike local nodal diprenorphine, saturating the area around the ganglion with diprenorphine did not alter the effect of systemically administered MEAP. These findings indicate that MEAP exerts its primary effect on vagal bradycardia by interacting with opiate receptors in the sinoatrial node and that parallel effects at the nearby parasympathetic ganglion are unlikely.
4. Discussion Our previous studies had demonstrated that systemic MEAP interrupted vagally mediated bradycardia but did not alter the bradycardia produced by the direct acting muscarinic agonist, methacholine. Those observations indicated that the administered opioid was not moderating the muscarinic receptor or its subsequent second messenger
system (Caffrey et al., 1995). The opiate receptors responsible for the vagolytic effect had to be located proximal to the pacemaker cells. The current data lead us to suggest that the sinoatrial node region appears to be the primary location where MEAP exerts its inhibition of vagal bradycardia. The fact that MEAP was able to inhibit vagal bradycardia when infused solely into the sinoatrial node confirms this region as a functional target for MEAP. Diprenorphine was able to reverse the effect of MEAP when they were both combined in the dialysis probe. This later observation reinforces the hypothesis that local nodal opiate receptors were responsible for the observed effect of MEAP. Systemic infusions of MEAP successfully inhibited vagal bradycardia as reported earlier (Caffrey et al., 1995; Caffrey, 1999). Although not statistically different from MEAP infused locally by microdialysis, inhibition of the vagus by MEAP may have been slightly more effective following systemic infusion. This observation suggests that the MEAP delivered by microdialysis may not have reached all of the nodal opiate receptors in every case. By comparison, nodal diprenorphine was equally effective versus both nodal and systemic MEAP. This may reflect differences in the relative diffusion or metabolic stability of the two compounds in the cardiac interstitium. The effect of systemic MEAP was eliminated by the nodal application of diprenorphine. Systemic infusions of MEAP, while under nodal blockade with diprenorphine resulted in vagal responses similar to those observed under control conditions. This suggests that there were few, if any, participating, opiate receptors located in the intracar-
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M. Farias et al. / Autonomic Neuroscience: Basic and Clinical 87 (2001) 9 – 15
diac parasympathetic ganglia. If a significant number of opiate receptors were concentrated in the ganglion, systemic MEAP should have interrupted vagal ganglionic transmission prior to and independent of the nodal blockade with diprenorphine. Since we demonstrated earlier that MEAP had no effect on the nodal response to methacholine, we presumed its effect was pre-junctional or ganglionic. When these new observations are included, one can empirically rule out a ganglionic site as the functional target. The possibility of ganglionic cells in the sinoatrial mediating this effect also seem unlikely although the possibility cannot be ruled out. Functional and histological mapping studies of the canine sinoatrial node report that the ganglion cells are localized to a fat pad overlying the right pulmonary vein just distal to but not in the sinoatrial node area (Bluemel et al., 1990). Nodal ganglion cells, if they existed, would also have to respond to MEAP differently from their nearby partners in the fat pad. This too seems unlikely. The opiate receptors in question are most likely located pre-junctionally on vagal nerve endings where the receptors suppress the release of acetylcholine within the sinoatrial node. The participation by opiate receptors on other nodal cell types (e.g. intrinsic cardiac neurons), capable of inhibiting the release of acetylcholine by another means, cannot be completely ruled out with the current data. For example, an opiate receptor mediated increase in the local norepinephrine concentration might reduce the release of acetylcholine through a pre-junctional a-adrenergic receptor. An a-adrenergic receptor mechanism would however also seem unlikely, since such a mechanism would be inconsistent with reports that effects related to those observed here were resistant to a-adrenergic blockade (Koyanagawa et al., 1989; Musha et al., 1989). Whether direct or indirect, the peptides most likely exert their effect by reducing the release of acetylcholine. This hypothesis is supported by the observation that enkephalin reduced the release of tritiated acetylcholine from rabbit atria pre-labeled in vitro with tritiated choline (Weitzell et al., 1984). The reduction in vagal bradycardia produced by MEAP is also unlikely to be mediated by a compensatory increase in b-adrenergic activation of the nodal pacemaker cells during vagal stimulation. In previous studies, the effect of MEAP was unaltered by prior b-adrenergic blockade (Caffrey et al., 1995) The heart is under the constant control of parasympathetic and sympathetic influences and cardiac opioids may play a crucial role in moderating these interactions. This suggestion is supported by the large abundance of proenkephalin mRNA found in the rat heart (Howells et al., 1986) and reports that sympathetic stimulation can produce enkephalin like effects on vagal bradycardia when the beta receptors are blocked (Koyanagawa et al., 1989). Similarities between the ability of MEAP and the sympathetic stimulation to inhibit vagal bradycardia, suggests that the sympathetic nervous system may release or provoke the
release of MEAP to govern the intensity of vagal responses. In summary the current results suggest that cardiac enkephalins regulate vagal bradycardia via opiate receptors that are concentrated within the sinoatrial node and not in the adjacent intracardiac parasympathetic ganglia. Finally, we suggest that these opiate receptors are probably located on efferent vagal nerve terminals within the node and that they exert their effect by reducing the release of acetylcholine.
Acknowledgements Martin Farias was supported by NIH Grant NIGMS GM51788. The authors would also like to thank Dr David Van Wylen, St. Olaf College, St. Olaf, Minnesota for technical advice and assistance with the fabrication of the dialysis probes used in these studies.
References Barron, B.A., Gaugl, J.F., Gu, H., Caffrey, J.L., 1992. Screening for opioids in the dog heart. J. Mol. Cell. Cardiol. 24, 67–77. Bluemel, K.M., Wurster, R.D., Randall, W.C., Duff, M.J., Otoole, M.F., 1990. Parasympathetic postganglionic pathways to the sinoatrial node. Am. J. Physiol. 259, H1504–H1510. Caffrey, J.L., Mateo, Z., Napier, L.D., Gaugl, J.F., Barron, B.A., 1995. Intrinsic cardiac enkephalins inhibit vagal bradycardia in the dog (Heart Circ Physiol 37). Am. J. Physiol. 268, H848–H855. Caffrey, J.L., 1999. Enkephalin Inhibits vagal control of heart, contractile force, and coronary blood flow in the dog in vivo. J. Auton. Nerv. Syst. 2318, 1–8. Howells, R.D., Kilpatrick, D.L., Bailey, L.C., Noe, M., Udenfriend, S., 1986. Proenkephalin mRNA in rat heart. Proc. Natl. Acad. Sci. USA 83, 1960–1963. Koyanagawa, H., Musha, T., Kanda, A., Kimura, T., Satoh, S., 1989. Inhibition of vagal transmission by cardiac sympathetic nerve stimulation in the dog: possible involvement of opioid receptor. J. Pharmacol. Exp. Ther. 250, 1092–1096. Low, K.G., Allen, R.G., Melner, M.H., 1990. Association of proenkephalin transcripts with polyribosomes in the heart. Mol. Endocrinol. 4, 1408–1415. Mateo, Z., Napier, L.D., Gaugl, J.F., Barron, B.A., Caffrey, J.L., 1995. Hemorrhage alters plasma and cardiac enkephalins and catelcholamines in anesthetized dogs (Heart Circ. Physiol. 38). Am. J. Physiol. 269, H2082–H2089. Musha, T., Satoh, E., Koyanagawa, H., Kimura, T., Satoh, S., 1989. Effects of opioid agonists on sympathetic and parasympathetic transmission to the dog heart. J. Pharmacol. Exp. Ther. 250, 1087– 1091. Pokrovsky, V.M., Oadchiy, O., 1995. Regulatory peptides as modulators of vagal influence on cardiac rhythm. Can. J. Physiol. Pharnmacol. 73, 1235–1245. Sadee, W., Pfeiffer, A., Herz, A., 1982. Opiate receptor: multiple effects of metal ions. J. Neurochem. 39, 659–667. Springhorn, J.P., Claycomb, W.C., 1989. Preproenkephalin mRNA expression in developing rat heart and in cultured ventricular cardiac muscle cells. Biochem. J. 258, 73–78. Springhorn, J.P., Claycomb, W.C., 1992. Translation of heart preproen-
M. Farias et al. / Autonomic Neuroscience: Basic and Clinical 87 (2001) 9 – 15 kephalin mRNA and the secretion of enkephalin containing peptides from cultured cardiac myocytes (Heart Circ Physiol 32). Am. J. Physiol. 263, H1560–H1565. Van Wylen, L D.G., Willis, J., Sodhi, J., Weiss, R.J., Lasley, R.D., Mentzer, R.M., 1990. Cardiac microdialysis to estimate interstitial
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adenosine and coronary blood flow (Heart Circ Physiol 27). Am. J. Physiol. 258, H1642–H1649. Weitzell, l R., Illes, P., Starke, K., 1984. Inhibition via opioid m- and d-receptors of vagal transmission in rabbit isolated heart. Arch. Pharmacol. 328, 186–190.
Local opiate receptors in the sinoatrial node moderate vagal bradycardia a b b b, Martin Farias , Keith Jackson , Amber Stanfill , James L. Caffrey * a
b
Department of Biology, University of Texas /Brownsville, Brownsville, TX 78520, USA Department of Integrative Physiology, Cardiovascular Research Institute, University of North Texas Health Science Center, UNTHSC-FW, 3500 Camp Bowie Boulevard, Fort Worth, TX 76106, USA Received 26 June 2000; received in revised form 6 September 2000; accepted 8 September 2000
Abstract Met-enkephalin-arg-phe (MEAP) interrupts vagal bradycardia when infused into the systemic circulation. This study was designed to locate the opiate receptors functionally responsible for this inhibition. Previous observations suggested that the receptors were most likely located in either intracardiac parasympathetic ganglia or the pre-junctional nerve terminals innervating the sinoatrial node. In this study 10 dogs were instrumented with a microdialysis probe inserted into the sinoatrial node. The functional position of the probe was tested by briefly introducing norepinephrine into the probe producing an increase in heart rate of more than 30 beats / min. Vagal stimulations were conducted at 0.5, 1,2 and 4 Hz during vehicle infusion (saline ascorbate). Cardiovascular responses during vagal stimulation were recorded on-line. MEAP was infused directly into the sinoatrial node via the microdialysis probe. The evaluation of vagal bradycardia was repeated during the nodal application of MEAP, diprenorphine (opiate antagonist), and diprenorphine co-infused with MEAP. MEAP introduced into the sinoatrial node via the microdialysis probe reduced vagal bradycardia by more than half. Simultaneous local nodal blockade of these receptors with the opiate antagonist, diprenorphine, eliminated the effect of MEAP demonstrating the participation by opiate receptors. Systemic infusions of MEAP produced a reduction in vagal bradycardia nearly identical to that observed during nodal administration. When local nodal opiate receptors were blocked with diprenorphine, the systemic effect of MEAP was eliminated. These data lead us to suggest that the opiate receptors responsible for the inhibition of vagal bradycardia are located within the sinoatrial node with few, if any, participating extra-nodal or ganglionic receptors. 2001 Elsevier Science B.V. All rights reserved. Keywords: Enkephalins; Sinoatrial node; Bradycardia; Vagus nerve; Microdialysis
1. Introduction Although the myocardium contains a variety of opioids, their function in heart is not well understood. Howells et al. (1986) originally found more proenkephalin mRNA in rat heart than in comparable samples of rat brain or adrenal gland. Despite rather limited concentrations of the fully processed met- and leu-enkephalin in myocardial extracts, the abundant message strongly suggested that the heart produced enkephalin. This hypothesis was subsequently confirmed by several laboratories who demonstrated that the RNA was translated and the product secreted in vitro (Springhorn and Claycomb, 1989; Low et al., 1990; Springhorn and Claycomb, 1992). In addition, large concentrations of immunoreactivity corresponding to intact proenkephalin and its C-terminal fragments, Peptide-B and *Corresponding author. Tel.: 11-817-735-2085; fax: 11-817-7355084. E-mail address: [email protected] (J.L. Caffrey).
met-enkephalin-arg-phe (MEAP), were identified in the dog heart (Barron et al., 1992; Mateo et al., 1995). Weitzell et al. (1984) initially demonstrated that enkephalins inhibited vagal transmission in isolated hearts. They reported that morphine, met-enkephalin, and D-Ala 2 , 5 D-Leu -enkephalin all reduced vagal bradycardia in rabbit hearts and the effect of each was reversed by the opiate antagonist, naloxone (Weitzell et al., 1984). Enkephalins have also been reported to suppress vagally induced bradycardia in vivo and may therefore function as governors of vagal control (Musha et al., 1989; Caffrey et al., 1995; Pokrovsky and Oadchiy, 1995; Caffrey, 1999). During adrenergic receptor blockade, a reduction in vagal bradycardia was observed when cardiac sympathetic nerves were activated. This vagolytic effect was reversed in part by the opiate antagonist naloxone. Since enkephalin produced a similar vagal inhibition, the authors suggested the impaired vagal function resulted from endogenous opiates (Koyanagawa et al., 1989). Our laboratory reported that MEAP was more effective than met-enkephalin in
1566-0702 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S1566-0702( 00 )00244-7
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M. Farias et al. / Autonomic Neuroscience: Basic and Clinical 87 (2001) 9 – 15
dogs and effectively attenuated vagally induced bradycardia when infused systemically at picomolar rates (Caffrey et al., 1995). Diprenorphine inhibited the effect of MEAP and provided support for participation by opiate receptors. We also have been able to demonstrate that MEAP can inhibit vagal control of heart rate, contractile force and coronary blood flow (Caffrey, 1999). The fact that enkephalins can inhibit vagal bradycardia appears well supported by the prior studies. The location of the opioid receptor that mediates this effect, however, remains unclear. MEAP did not alter the negative chronotropic effect of the direct acting, muscarinic agonist, methacholine (Caffrey et al., 1995). The inability to alter the response to methacholine suggested that MEAP exerted its effect at a site in the efferent vagal tract that is proximal to the nodal, muscarinic receptors. This narrows the location of these opiate receptors to sites within the intracardiac parasympathetic ganglia or at prejunctional sites (e.g. vagal nerve terminals) within the sinoatrial node (Caffrey et al., 1995). Since the two primary candidates, the sinoatrial node and the intracardiac parasympathetic ganglia are anatomically adjacent to one another and the sinoatrial node artery perfuses both, unambiguously assigning target status to one or both has been technically difficult. The following study makes an innovative use of microdialysis to more precisely locate the opioid receptors responsible for the ability of MEAP to modify vagal bradycardia.
2. Materials and methods Ten dogs were assigned to various experimental protocols. All protocols were approved by the Institutional Animal Care and Use Committee and were in compliance with the NIH Guide for the care and use of laboratory animals. The dogs were anesthetized with pentobarbital sodium (32.5 mg / kg). The dogs were intubated and mechanically ventilated initially at 225 ml / min per kg with room air. Fluid filled catheters were then inserted into the right femoral artery and vein and advanced respectively into the descending aorta and inferior vena cava. The arterial line was attached to a Statham PD23XL pressure transducer and heart rates were measured continuously on-line (MacLabs). The venous line was positioned above the diaphragm and was used to administer supplemental anesthetic and to infuse peptides systemically. The acid– base balance and the blood gases were determined regularly with an Instrumentation Laboratories Blood Gas Analyzer. The pO 2 (90–120 mmHg), the pH (7.35–7.45) and the pCO 2 (30–40 mmHg) were adjusted to normal by administering supplemental oxygen, bicarbonate or modifying the minute volume. Supplemental anesthetic was administered intravenously as required. The right and left vagus nerves were isolated through a midline surgical incision and ligated tightly with umbilical
tape for later retrieval. Surgical anesthesia was carefully monitored, and a single dose of succinylcholine (50 mg / kg) was administered intravenously to temporarily reduce involuntary movements of the muscles during the 10–15 min required for electrosurgical incision of the chest and removal of the right ribs two to five. The heart was exposed from the right aspect. The pericardium was opened and the dorsal pericardial margins were sutured to the body wall to provide a pericardial cradle. A 25 g stainless steel spinal needle containing a microdialysis line was inserted into the center of the long axis of the sinoatrial node. The needle was removed and the probe was then positioned so that the dialysis window was within the substance of the sinoatrial node. The microdialysis probes were constructed of a single 1 cm length of dialysis fiber derived from a Clirans 10 (Terumo Medical Corporation, Sumerset, NJ) artificial kidney. The dialysis tubing had a 300 mm inner diameter (ID), a 305 mm outer diameter (OD) and a molecular weight cutoff of 5000. The inflow and outflow lines were composed of hollow silica tubing (SGE, Austin, TX) glued into the dialysis fiber (Van Wylen et al., 1990). Norepinephrine (1 nmol / ml) was diluted in saline and 0.1% ascorbic acid (vehicle) and perfused into the microdialysis probe at 4 ml / min in order to confirm functionally the position of the probe in the sinoatrial node. If properly positioned, the heart rate routinely increased by 30 beats / min (bpm) or more within 30 s. This tachycardia was not observed when norepinephrine was introduced into probes placed elsewhere in the atrial wall. The norepinephrine was flushed out and the probe was perfused with vehicle for 60 min. Vehicle or peptide was then perfused through the probe while the right vagus nerve was stimulated at 0.5 Hz for 15 s at a supra-maximal voltage (usually 15 V). The system was allowed 1 min and 45 s to recover. A complete frequency response curve was then constructed by repeating the 2-min stimulus / recovery protocol sequentially at 1, 2, and 4 Hz. Fifteen minutes were subsequently allowed to elapse between each series of frequency responses. When employed, systemic peptide infusions were administered intravenously at 3 nmol / min per kg. This infusion rate was previously determined to provide the maximal inhibition of the vagal control of heart rate. All peptides were prepared in saline with 0.1% ascorbic acid to prevent oxidation and were infused for 5 min prior to beginning any stimulation protocols.
2.1. Protocol I: Intranodal MEAP and intranodal diprenorphine This initial protocol consisted of four treatments designed to determine if local opiate receptors in the sinoatrial node were responsible for the MEAP mediated inhibition of vagal bradycardia. The experiment was also designed to verify that opiate receptors were in fact responsible for this effect. All treatments were infused at
M. Farias et al. / Autonomic Neuroscience: Basic and Clinical 87 (2001) 9 – 15
4ul / min into the substance of the sinoatrial node using a micro-syringe pump. Saline ascorbate served as vehicle control for comparison with MEAP (1 nmol / ml), the opiate antagonist diprenorphine (0.22 nmol / ml) and the co-infusion of both agonist and antagonist together. MEAP was selected for these studies because it is found in high concentrations in the dog heart, it is a natural agonist, and it is more effective than methionine enkephalin (Barron et al., 1992; Caffrey et al., 1995; Mateo et al., 1995). Since we were unsure of which opiate receptor might be responsible for the expected effects, diprenorphine was chosen as the opiate antagonist in these studies because of its high affinity and relative non-selectivity at each of the three opiate receptor subtypes (Sadee et al., 1982).
2.2. Protocol II: Systemic MEAP and intranodal diprenorphine This protocol was designed to determine if the intracardiac parasympathetic ganglia also served as effective targets where MEAP could modify vagal bradycardia. The four treatments from Protocol I were repeated to verify that the nodal diprenorphine applied was adequate to block nodal opiate receptors. MEAP (3 nmol / min per kg) was then infused systemically to compare its effect with nodal administration. Prior studies have narrowed down the likely location of the opiate receptors responsible for the inhibition of bradycardia to the sinoatrial node and / or the intracardiac parasympathetic ganglia. As a result, MEAP was also administered intravenously while local nodal opiate receptors were blocked with the opiate receptor antagonist diprenorphine (0.22 nmol / ml) delivered via the microdialysis probe.
2.3. Materials Methionine enkephalin-arg-phe was synthesized by American Peptide Co, Sunnyvale, CA and diprenorphine was obtained from the National Institutes for Drug Abuse. All microdialysis probes were fabricated by David G.L. Van Wylen of St. Olaf College in St. Olaf Minnesota.
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2.4. Statistics The data are expressed throughout as means and standard errors. Differences were evaluated with an analysis of variance and multiple post-hoc comparisons were made with the aid of Tukey’s protected-t (GB-STATE, Dynamic Microsystems, Silver Spring, MD). Repeated measures designs were used where appropriate. P,0.05 was accepted as statistically different.
3. Results Table 1 provides the resting cardiovascular parameters for all animals across all treatments. As indicated there were no changes in baseline function prior to stimulation. Fig. 1 provides a schematic representation of the spatial relationships among the ganglion cells, the sinoatrial (SA) node, and their common vascular supply, the SA node artery. The SA node in the average 20 kg dog appears to be approximately 10–20 mm long and 2–3 mm in diameter. The structures are not drawn to scale and the fat pad is much larger than the node. In order to verify the functional position of the nodal microdialysis probe, norepinephrine (100 ng / ml) was infused briefly at 4 ml / min. A rapid increase in heart rate between 30 and 60 beats was routinely observed and was considered sufficient evidence of optimal positioning of the probe. If norepinephrine was introduced into probes placed elsewhere in the atrial free wall no comparable increase in heart rate was observed Fig. 2 illustrates the change in heart rate in 10 dogs during vagal stimulations at 0.5, 1, 2, and 4 Hz under the four different treatments of Protocol I. Infusion of saline ascorbate into the nodal probe had no effect on the resting heart rate. Stimulation of the right vagus nerve produced average declines in heart rate of 13, 21, 35 and 67 bpm, respectively. After 15 min, MEAP was introduced into the probe and perfused for an additional 5 min prior to stimulation. MEAP had no effect on the resting heart rate. The subsequent vagally induced bradycardia however, was reduced by more than one-half of that observed under
Table 1 Resting cardovascular indices
Heart rate (bpm) MAP (mmHg) Systolic (mmHg) Diastolic (mmHg)
Control
Nodal MEAP
13867
13067
11666
Nodal diprenorphine
Nodal MEAP nodal diprenorphine
Systemic MEAP
Systemic MEAP nodal diprenorphine
12869
13467
134610
143610
11466
11467
10966
10968
11468
14468
14268
140610
13668
133611
138611
10465
10165
10267
9765
9768
10168
12
M. Farias et al. / Autonomic Neuroscience: Basic and Clinical 87 (2001) 9 – 15
Fig. 1. Schematic representation of the spatial relationships in the area of the SA node. The structures are not drawn to scale.
control conditions. With MEAP in the dialysis probe, stimulation of the vagus nerve produced graded declines in heart rate of 6, 10, 17, and 28 bpm. After flushing the probe with saline, the vagal response routinely returned to normal within 5 min. Fifteen minutes after flushing out the
Fig. 2. Changes in heart rate mediated by right vagal stimulation under control conditions during vehicle applied by microdialysis (d), during nodal MEAP applied by microdialysis (j), after nodal opiate receptor blockade with diprenorphine applied by microdialysis probe (m), and MEAP1diprenorphine applied by microdialysis probe (.). Values are means; error bars have been omitted for clarity; n58–10. * Significantly different from the three lower curves at the same frequency.
peptide, the opiate antagonist, diprenorphine was introduced into the probe. Diprenorphine had no effect on the resting heart rate and the heart rate response during vagal stimulation was not statistically different from that observed under control conditions. When MEAP and diprenorphine were co-infused into the sinoatrial node, there was again no effect on the resting heart rate. Stimulation of the right vagus nerve during the combined treatment produced responses that were not statistically different from the control stimulations. Since local diprenorphine blocked the effect of local MEAP, the effect of MEAP must be mediated by nodal opiate receptors. The reduced vagal bradycardia produced by MEAP applied by microdialysis to the node was statistically different from the other three treatments in this protocol. Fig. 3 illustrates the change in heart rate for six dogs with two additional treatments. The first four treatments are the same as in protocol one. The additional treatments included systemic MEAP (3 nmol / min per kg, iv) and systemic MEAP combined with local diprenorphine administered by dialysis into at the sinoatrial node. These two treatments had no effect on resting heart. The results from the first four treatments were the same as those in Fig. 1, but were repeated to provide a direct comparison to the earlier experiments and to verify that the dose of diprenorphine was sufficient to block the nodal effect of MEAP. The dose of MEAP used systemically was previously determined to produce the maximal inhibition of vagal bradycardia (Caffrey et al., 1995). Systemic MEAP infusions reduced vagal bradycardia to an extent similar to that observed after the nodal application of the peptide. However, when diprenorphine was reintroduced into the nodal probe the vagolytic effect of systemic MEAP was
M. Farias et al. / Autonomic Neuroscience: Basic and Clinical 87 (2001) 9 – 15
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Fig. 3. Changes in heart rate mediated by right vagal stimulation under control conditions (d), during intravenous MEAP infusion (j), and during intravenous MEAP combined with nodal opiate receptor blockade produced by diprenorphine applied by microdialysis (m). Values are means; error bars have been omitted for clarity; n56–10. * Significantly different from the two lower curves at the same frequency.
completely eliminated. During this nodal opiate receptor blockade, vagal stimulations produced graded declines in heart rate indistinguishable from control. Although unlikely, we considered the possibility that nodal diprenorphine might also reach the nearby parasympathetic ganglion cells by retrograde diffusion along nerve tracks between the ganglion and the SA node. To test this hypothesis, the systemic effect of MEAP was evaluated in two dogs before and after infiltrating the ganglionic fat pad with diprenorphine. The dose of diprenorphine was equal to the entire content perfused through the probe during each of the protocol II experiments described above. Under these circumstances, the vagolytic effect of systemic MEAP was identical before and after the ganglionic administration of diprenorphine. Unlike local nodal diprenorphine, saturating the area around the ganglion with diprenorphine did not alter the effect of systemically administered MEAP. These findings indicate that MEAP exerts its primary effect on vagal bradycardia by interacting with opiate receptors in the sinoatrial node and that parallel effects at the nearby parasympathetic ganglion are unlikely.
4. Discussion Our previous studies had demonstrated that systemic MEAP interrupted vagally mediated bradycardia but did not alter the bradycardia produced by the direct acting muscarinic agonist, methacholine. Those observations indicated that the administered opioid was not moderating the muscarinic receptor or its subsequent second messenger
system (Caffrey et al., 1995). The opiate receptors responsible for the vagolytic effect had to be located proximal to the pacemaker cells. The current data lead us to suggest that the sinoatrial node region appears to be the primary location where MEAP exerts its inhibition of vagal bradycardia. The fact that MEAP was able to inhibit vagal bradycardia when infused solely into the sinoatrial node confirms this region as a functional target for MEAP. Diprenorphine was able to reverse the effect of MEAP when they were both combined in the dialysis probe. This later observation reinforces the hypothesis that local nodal opiate receptors were responsible for the observed effect of MEAP. Systemic infusions of MEAP successfully inhibited vagal bradycardia as reported earlier (Caffrey et al., 1995; Caffrey, 1999). Although not statistically different from MEAP infused locally by microdialysis, inhibition of the vagus by MEAP may have been slightly more effective following systemic infusion. This observation suggests that the MEAP delivered by microdialysis may not have reached all of the nodal opiate receptors in every case. By comparison, nodal diprenorphine was equally effective versus both nodal and systemic MEAP. This may reflect differences in the relative diffusion or metabolic stability of the two compounds in the cardiac interstitium. The effect of systemic MEAP was eliminated by the nodal application of diprenorphine. Systemic infusions of MEAP, while under nodal blockade with diprenorphine resulted in vagal responses similar to those observed under control conditions. This suggests that there were few, if any, participating, opiate receptors located in the intracar-
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M. Farias et al. / Autonomic Neuroscience: Basic and Clinical 87 (2001) 9 – 15
diac parasympathetic ganglia. If a significant number of opiate receptors were concentrated in the ganglion, systemic MEAP should have interrupted vagal ganglionic transmission prior to and independent of the nodal blockade with diprenorphine. Since we demonstrated earlier that MEAP had no effect on the nodal response to methacholine, we presumed its effect was pre-junctional or ganglionic. When these new observations are included, one can empirically rule out a ganglionic site as the functional target. The possibility of ganglionic cells in the sinoatrial mediating this effect also seem unlikely although the possibility cannot be ruled out. Functional and histological mapping studies of the canine sinoatrial node report that the ganglion cells are localized to a fat pad overlying the right pulmonary vein just distal to but not in the sinoatrial node area (Bluemel et al., 1990). Nodal ganglion cells, if they existed, would also have to respond to MEAP differently from their nearby partners in the fat pad. This too seems unlikely. The opiate receptors in question are most likely located pre-junctionally on vagal nerve endings where the receptors suppress the release of acetylcholine within the sinoatrial node. The participation by opiate receptors on other nodal cell types (e.g. intrinsic cardiac neurons), capable of inhibiting the release of acetylcholine by another means, cannot be completely ruled out with the current data. For example, an opiate receptor mediated increase in the local norepinephrine concentration might reduce the release of acetylcholine through a pre-junctional a-adrenergic receptor. An a-adrenergic receptor mechanism would however also seem unlikely, since such a mechanism would be inconsistent with reports that effects related to those observed here were resistant to a-adrenergic blockade (Koyanagawa et al., 1989; Musha et al., 1989). Whether direct or indirect, the peptides most likely exert their effect by reducing the release of acetylcholine. This hypothesis is supported by the observation that enkephalin reduced the release of tritiated acetylcholine from rabbit atria pre-labeled in vitro with tritiated choline (Weitzell et al., 1984). The reduction in vagal bradycardia produced by MEAP is also unlikely to be mediated by a compensatory increase in b-adrenergic activation of the nodal pacemaker cells during vagal stimulation. In previous studies, the effect of MEAP was unaltered by prior b-adrenergic blockade (Caffrey et al., 1995) The heart is under the constant control of parasympathetic and sympathetic influences and cardiac opioids may play a crucial role in moderating these interactions. This suggestion is supported by the large abundance of proenkephalin mRNA found in the rat heart (Howells et al., 1986) and reports that sympathetic stimulation can produce enkephalin like effects on vagal bradycardia when the beta receptors are blocked (Koyanagawa et al., 1989). Similarities between the ability of MEAP and the sympathetic stimulation to inhibit vagal bradycardia, suggests that the sympathetic nervous system may release or provoke the
release of MEAP to govern the intensity of vagal responses. In summary the current results suggest that cardiac enkephalins regulate vagal bradycardia via opiate receptors that are concentrated within the sinoatrial node and not in the adjacent intracardiac parasympathetic ganglia. Finally, we suggest that these opiate receptors are probably located on efferent vagal nerve terminals within the node and that they exert their effect by reducing the release of acetylcholine.
Acknowledgements Martin Farias was supported by NIH Grant NIGMS GM51788. The authors would also like to thank Dr David Van Wylen, St. Olaf College, St. Olaf, Minnesota for technical advice and assistance with the fabrication of the dialysis probes used in these studies.
References Barron, B.A., Gaugl, J.F., Gu, H., Caffrey, J.L., 1992. Screening for opioids in the dog heart. J. Mol. Cell. Cardiol. 24, 67–77. Bluemel, K.M., Wurster, R.D., Randall, W.C., Duff, M.J., Otoole, M.F., 1990. Parasympathetic postganglionic pathways to the sinoatrial node. Am. J. Physiol. 259, H1504–H1510. Caffrey, J.L., Mateo, Z., Napier, L.D., Gaugl, J.F., Barron, B.A., 1995. Intrinsic cardiac enkephalins inhibit vagal bradycardia in the dog (Heart Circ Physiol 37). Am. J. Physiol. 268, H848–H855. Caffrey, J.L., 1999. Enkephalin Inhibits vagal control of heart, contractile force, and coronary blood flow in the dog in vivo. J. Auton. Nerv. Syst. 2318, 1–8. Howells, R.D., Kilpatrick, D.L., Bailey, L.C., Noe, M., Udenfriend, S., 1986. Proenkephalin mRNA in rat heart. Proc. Natl. Acad. Sci. USA 83, 1960–1963. Koyanagawa, H., Musha, T., Kanda, A., Kimura, T., Satoh, S., 1989. Inhibition of vagal transmission by cardiac sympathetic nerve stimulation in the dog: possible involvement of opioid receptor. J. Pharmacol. Exp. Ther. 250, 1092–1096. Low, K.G., Allen, R.G., Melner, M.H., 1990. Association of proenkephalin transcripts with polyribosomes in the heart. Mol. Endocrinol. 4, 1408–1415. Mateo, Z., Napier, L.D., Gaugl, J.F., Barron, B.A., Caffrey, J.L., 1995. Hemorrhage alters plasma and cardiac enkephalins and catelcholamines in anesthetized dogs (Heart Circ. Physiol. 38). Am. J. Physiol. 269, H2082–H2089. Musha, T., Satoh, E., Koyanagawa, H., Kimura, T., Satoh, S., 1989. Effects of opioid agonists on sympathetic and parasympathetic transmission to the dog heart. J. Pharmacol. Exp. Ther. 250, 1087– 1091. Pokrovsky, V.M., Oadchiy, O., 1995. Regulatory peptides as modulators of vagal influence on cardiac rhythm. Can. J. Physiol. Pharnmacol. 73, 1235–1245. Sadee, W., Pfeiffer, A., Herz, A., 1982. Opiate receptor: multiple effects of metal ions. J. Neurochem. 39, 659–667. Springhorn, J.P., Claycomb, W.C., 1989. Preproenkephalin mRNA expression in developing rat heart and in cultured ventricular cardiac muscle cells. Biochem. J. 258, 73–78. Springhorn, J.P., Claycomb, W.C., 1992. Translation of heart preproen-
M. Farias et al. / Autonomic Neuroscience: Basic and Clinical 87 (2001) 9 – 15 kephalin mRNA and the secretion of enkephalin containing peptides from cultured cardiac myocytes (Heart Circ Physiol 32). Am. J. Physiol. 263, H1560–H1565. Van Wylen, L D.G., Willis, J., Sodhi, J., Weiss, R.J., Lasley, R.D., Mentzer, R.M., 1990. Cardiac microdialysis to estimate interstitial
15
adenosine and coronary blood flow (Heart Circ Physiol 27). Am. J. Physiol. 258, H1642–H1649. Weitzell, l R., Illes, P., Starke, K., 1984. Inhibition via opioid m- and d-receptors of vagal transmission in rabbit isolated heart. Arch. Pharmacol. 328, 186–190.