Keith E. Jackson

Local opiate receptors in the sinoatrial node moderate vagal bradycardia.

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.

Autonomic control of heart rate in dogs treated chronically with morphine

The vagotonic effect of chronic morphine on the parasympathetic control of the heart was examined in dogs treated with morphine for 2 wk. Because normal vagal function is critical to myocardial stability, the study was conducted to evaluate for potential impairments following chronic vagal stimulation. The hypothesis that persistent vagal outflow would result in a loss of vagal reserve and reduced vagal control of heart rate was tested. Heart rate and the high-frequency variation in heart rate (power spectral analysis) declined shortly after initiation of subcutaneous morphine infusion. A progressive bradycardia correlated well with the rising plasma morphine. The resting bradycardia (57 beats/min) was maintained through day 2 and was accompanied by a significant parallel increase in vagal effect and a decline in the intrinsic heart rate (160 vs. 182 beats/min). A compensatory increase in the ambient sympathetic control of heart rate was evident on day 2 and was supported by an increase in circulating catecholamines. The lowered intrinsic heart rate and elevated sympathetic activity were maintained through day 10 despite a return of the resting heart rate and plasma catecholamines to pretreatment values. These observations suggested that chronic morphine alters either the intrinsic function of the sinoatrial node or reduces the postvagal tachycardia normally attributed to nonadrenergic, noncholinergic agents. Both acute and chronic morphine depressed the rate of development of bradycardia during direct vagal nerve stimulation without altering the rate of recovery afterward. This last observation suggests that acute morphine reduces the rate of acetylcholine release. Results provide insight into the mechanisms that maintain vagal responsiveness. The results are also relevant clinically because opiates are increasingly prescribed for chronic pain and opiate abuse is currently in resurgence.The vagotonic effect of chronic morphine on the parasympathetic control of the heart was examined in dogs treated with morphine for 2 wk. Because normal vagal function is critical to myocardial stability, the study was conducted to evaluate for potential impairments following chronic vagal stimulation. The hypothesis that persistent vagal outflow would result in a loss of vagal reserve and reduced vagal control of heart rate was tested. Heart rate and the high-frequency variation in heart rate (power spectral analysis) declined shortly after initiation of subcutaneous morphine infusion. A progressive bradycardia correlated well with the rising plasma morphine. The resting bradycardia (57 beats/min) was maintained through day 2 and was accompanied by a significant parallel increase in vagal effect and a decline in the intrinsic heart rate (160 vs. 182 beats/min). A compensatory increase in the ambient sympathetic control of heart rate was evident on day 2 and was supported by an increase in circulating catecholamines. The lowered intrinsic heart rate and elevated sympathetic activity were maintained through day 10 despite a return of the resting heart rate and plasma catecholamines to pretreatment values. These observations suggested that chronic morphine alters either the intrinsic function of the sinoatrial node or reduces the postvagal tachycardia normally attributed to nonadrenergic, noncholinergic agents. Both acute and chronic morphine depressed the rate of development of bradycardia during direct vagal nerve stimulation without altering the rate of recovery afterward. This last observation suggests that acute morphine reduces the rate of acetylcholine release. Results provide insight into the mechanisms that maintain vagal responsiveness. The results are also relevant clinically because opiates are increasingly prescribed for chronic pain and opiate abuse is currently in resurgence.

Transient arterial occlusion raises enkephalin in the canine sinoatrial node and improves vagal bradycardia

The C-terminal proenkephalin sequence, Methionine-enkephalin-arginine-phenylalanine (MEAP), is abundant in the myocardium and when delivered into the sinoatrial (SA) node by microdialysis, the peptide had significant vagolytic activity. The study that follows was conducted to determine if an increase in endogenous nodal MEAP could be demonstrated during reduced nodal blood flow and was endogenous MEAP similarly vagolytic. Microdialysis probes were placed in the canine SA node and perfused at 5 microl per min. The SA node artery was occluded and released four times at 10-min intervals. The intermittent occlusions were followed by one or two prolonged occlusions (30 min). Vagally mediated bradycardia was compared before, during, and after occlusion of the artery. An increase in recovered MEAP (70-220 fmol) was recorded during each of the initial 10-min occlusions. MEAP returned to baseline during each subsequent 10-min reperfusion. There was a sustained increase in MEAP (110-150 fmol) during longer occlusions. Contrary to the hypothesis, the increased MEAP during arterial occlusion was coincident with improved vagal bradycardia. The improvement in vagally mediated bradycardia was highly reproducible and was observed again during a second 30-min occlusion. The improved vagal function was reversed or reduced, respectively, when naltrindole or glibenclamide was included in the microdialysis inflow during arterial occlusion. Although these observations suggested that opioid receptors and ATP-sensitive K+ channels might have been involved, only a single dose of each agent was practical. Therefore, the specificity of these two responses remains to be confirmed. In summary, the recovery of endogenous opioids from the sinoatrial node increased during reduced arterial perfusion of the node. Contrary to expectations, the increase in recovered endogenous opioids was accompanied byimproved rather than impaired vagal bradycardia.

Delta Opioid Receptors Inhibit Vagal Bradycardia in the Sinoatrial Node

Background: Methionine-enkephalin-arginine-phenylalanine (MEAP) is an endogenous opiate derived from the C-terminal sequence of the larger precursor molecule proenkephalin. This heptapeptide is abundant in the myocardium and has significant vagolytic activity when infused systemically. MEAP interrupted vagal bradycardia when it was delivered directly into the sinoatrial node by local microdialysis. This study was conducted to determine the opioid receptor responsible for the vagolytic effect of MEAP. Methods and Results: Microdialysis probes were placed in the sinoatrial node of mongrel dogs and perfused at 5 μL/min. Increasing doses of MEAP were included in the nodal perfusate and approximately two thirds of the vagal bradycardia was inhibited with a maximal effect at 0.3 nmoles/μ,L and a half-maximal response near 0.1 nmoles/,uL. When deltorphin II (a delta opioid receptor agonist) was infused into the sinoatrial node, more than 95% of the vagal bradycardia was eliminated at 0.3 nmoles/,uL with the half-maximal response near 0.1 nmoles/,L, indicating that deltorphin II was more efficacious than MEAP. The maximal deltorphin II and MEAP effects were both similarly reversed by the paired infusion of increasing doses of the 6 opiate receptor antagonist, naltrindole. Selected, μ (endomorphin, super DALDA) and κ (dynorphin, U50488) receptor agonists and,μ (CTAP) and κ (norBNI) receptor antagonists were completely ineffective in this system. Conclusions: These data suggest that the vagolytic effect of MEAP involves the activation of delta opiate receptors within the sinoatrial node.

Leucine-enkephalin interrupts sympathetically mediated tachycardia prejunctionally in the canine sinoatrial node.

This study examined the role of leucine-enkephalin (LE) in the sympathetic regulation of the cardiac pacemaker. LE was administered by microdialysis into the interstitium of the canine sinoatrial node during either sympathetic nerve stimulation or norepinephrine infusion. In study one, the right cardiac sympathetic nerves were isolated as they exit the stellate ganglion and were stimulated to produce graded (low, 20–30 bpm; high 40–50 bpm) increases in heart rate (HR). LE (1.5 nmoles/min) was added to the dialysis inflow and the sympathetic stimulations were repeated after 5 and 20 min of LE infusion. After 5 min, LE reduced the tachycardia during sympathetic stimulation at both low (18.2 ± 1.3 bpm to 11.4 ± 1.4 bpm) and high (45 ± 1.5 bpm to 22.8 ± 1.5 bpm) frequency stimulations. The Inhibition was maintained during 20 min of continuous LE exposure with no evidence of opioid desensitization. The δ-opioid antagonist, naltrindole (1.1 nmoles/min), restored only 30% of the sympathetic tachycardia. Nodal δ-receptors are vagolytic and vagal stimulations were included in the protocol as positive controls. LE reduced vagal bradycardia by 50% and naltrindole completely restored the vagal bradycardia. In Study 2, additional opioid antagonists were used to determine if alternative opioid receptors might be implicated in the sympatholytic response. Increasing doses of the K-antagonist, norbinaltorphimine (norBNI), were combined with LE during sympathetic stimulation. NorBNI completely restored the sympathetic tachycardia with an ED50 of 0.01 nmoles/min. A single dose of the μ-antagonist, CTAP (1.0 nmoles/min), failed to alter the sympatholytic effect of LE. Study 3 was conducted to determine if the sympatholytic effect was prejunctional or postjunctional in character. Norepinephrine was added to the dialysis Inflow at a rate (30–45 pmoles/min) sufficient to produce intermediate increases (35.2 ± 1.8 bpm) in HR. LE was then combined with norepinephrine and responses were recorded at 5-min intervals for 20 min. The tachycardia mediated by added norepinephrine was unaltered by LE or LE plus naltrindole. At the same 5-min intervals, LE reduced vagal bradycardia by more than 50%. This vagolytic effect was again completely reversed by naltrindole. Collectively, these observations support the hypothesis that the local nodal sympatholytic effect of LE was mediated by κ-opioid receptors that reduced the effective interstitial concentration of norepinephrine and not the result of a postjunctional interaction between LE and norepinephrine.

Cardiac microdialysis a powerful tool

See article by Akiyama et al. [1] (pages 531–538) in this issue. Tsuyoshi Akiyama and Toji Yamazaki have reported that norepinephrine inhibits acetylcholine release from post-ganglionic cardiac vagal efferents [1]. This is hardly a novel idea since the existence of pre-junctional adrenergic receptors, presumably located on cardiac parasympathetic efferents, has been well documented both physiologically and pharmacologically. What is unique here is that the authors have actually measured steady-state changes in acetylcholine within the interstitium of the working left ventricle. They have done this with an elegant marriage of in situ microdialysis and an ultra-sensitive HPLC-electrochemical detection system. The report referenced above represents another step forward in the continuing struggle to evaluate the paracrine environment within the myocardium. Much of the early work in this regard was focused on quantifying the dynamics of capillary filtration including the osmotic contribution of interstitial constituents and the measurement of interstitial fluid pressure. Every student of physiology remembers the measurements of interstitial fluid pressure obtained by Guyton from porous spheres inserted into a variety of tissues [2]. Subsequent efforts to explain the regulation of coronary blood flow produced a variety of experimental incursions into the myocardial interstitium. Interstitial constituents were estimated from a variety of sources including micropipettes, electrodes, filter disks, lymph cannulas and pericardial perfusions. The controversies centered on the adenosine hypothesis were responsible for much of the later drive to investigate the myocardial interstitium. In this regard, Van Wylen et al. [3] reported the application of an existing technique, microdialysis, to estimate interstitial adenosine and its metabolites. Microdialysis, which evolved during the prior decade, had largely been limited to studies evaluating neurotransmitter release within the central nervous system. The active generation … * Corresponding author. Tel.: +1-817-735-2085; fax: +1-817-735-5084 jcaffrey{at}hsc.unt.edu

62 HEME-DERIVED CARBON MONOXIDE PROMOTES ENDOTHELIAL DYSFUNCTION AND HYPERTENSION IN OBESE ZUCKER RATS

Vascular heme oxygenase (HO) metabolizes heme to form carbon monoxide (CO). Heme-derived CO inhibits nitric oxide synthase and promotes endothelial dysfunction (ED) in salt-induced hypertension. The obese Zucker rat (ZR) is a model of type 2 diabetes showing hypertension and vascular ED. This study tests the hypothesis that HO-derived CO contributes to arteriolar ED and hypertension in obese ZR. Male obese (548 ± 12 g, n = 22) and lean (309 ± 14 g, n = 12) ZR were used for the study. Obese ZR had elevated mean arterial blood pressure (154 ± 3 mm Hg vs. 121 ± 4 mm Hg), blood glucose (186 ± 7 mg/dL vs. 140 ± 3 mg/dL) and blood carboxyhemoglobin (HbCO) levels (3.9 ± 0.1% vs. 3.0 ± 0.1%). Experiments used isolated skeletal muscle arterioles with constant (80 mm Hg) pressure and no flow, or constant midpoint, but altered endpoint pressures to establish graded levels of luminal flow (0-50 μL/min). In obese ZR arterioles, responses to an endothelium-dependent vasodilator, acetylcholine (ACh, 1 nmol/L - 3 μmol/L) (Δmax 32 ± 4 μm, n = 8 vs. 62 ± 7 μm, n = 5) and flow (Δmax-1 ± 1 μm, n = 4 vs. 21 ± 2 μm, n = 4) were attenuated. Acute in vitro pretreatment with a HO inhibitor, chromium mesoporphyrin (CrMP, 15 μmol/L), enhanced ACh (Δmax 59 ± 8 μm, n = 7 vs. 58 ± 13 μm, n = 5) and flow-induced dilation (Δmax 19 ± 2 μm, n = 4 vs. 20 ± 3 μm, n = 3) and abolished the differences between lean and obese ZR arterioles. Furthermore, exogenous CO (100 μmol/L) prevented the restoration of flow-induced dilation by the HO inhibitor (Δmax-1 ± 3 μm, n = 4) in obese ZR arterioles. In awake obese ZR instrumented with chronic femoral arterial catheters, administration of a HO inhibitor (25 μmol/kg/day zinc deuteroporphyrin 2,4-bisglycol IP for 3 days) lowered the blood pressure (151 ± 1 to 109 ± 1 mm Hg, n = 3). These data show that HbCO levels are increased, and arteriolar endothelium-dependent vasodilation is decreased in obese ZR. Acute in vitro treatment with a HO inhibitor restores endothelium-dependent responses to lean ZR levels, but exogenous CO prevents this effect. Furthermore, HO inhibition lowers blood pressure in obese ZR. These results suggest that heme-derived CO production is increased and contributes to arteriolar ED and hypertension in obese ZR, and hence identify vascular HO as a novel therapeutic target to prevent ED and hypertension in obesity and type 2 diabetes.

65 L-ARGININE RESTORES CORONARY ENDOTHELIAL FUNCTION IN OBESE ZUCKER RATS

The release of nitric oxide (NO) through the oxidation of L-arginine by endothelial NO synthase (eNOS) promotes relaxation of vascular smooth muscle and plays a pivotal role in the maintenance of vascular homeostasis. Since L-arginine is an exclusive substrate for eNOS, its availability can regulate NO formation. Endothelial dysfunction, due to decreased NO function, is a prominent feature of obesity and type 2 diabetes and contributes to cardiovascular pathology. While eNOS expression is unchanged, vascular NO levels are decreased and L-arginine administration lowers blood pressure in patients with type 2 diabetes. This study tests the hypothesis that L-arginine restores coronary arterial endothelial function in obese Zucker rats (ZR), experimental models of type 2 diabetes. Male obese ZR (548 ± 12 g) and lean Sprague-Dawley (SD) rats (300 g) were used for the study. Mean arterial blood pressure (154 ± 3 mm Hg) and fasting blood glucose levels (186 ± 7 mg/dL) were elevated in obese ZR. Experiments were performed on isolated pressurized (80 mm Hg no flow conditions) small septal coronary arteries (200-300 μm) superfused with oxygenated (14% O2, 5% CO2 balanced with N2) Krebs buffer. Responses to an endothelium- and NO-dependent vasodilator, acetylcholine (1 nmol/L-3 μmol/L) were greatly attenuated in obese ZR coronary vessels (Δmax 5 ± 2 μm vs. 54 ± 10 μm). Acute in vitro pretreatment with the NOS substrate, 1 mmol/L L-arginine, enhanced acetylcholine-induced dilation and abolished the differences between obese ZR and SD arteries (Δmax 48 ± 18 μm vs. 61 ± 18 μm). These data show that blood pressure and blood glucose levels are elevated in obese ZR. Acetylcholine-induced endothelium-dependent vasodilation is greatly attenuated in small coronary arteries isolated from obese ZR. In addition, acute in vitro administration of L-arginine enhanced coronary endothelium-dependent responses and abolished the differences between obese ZR and SD coronary vessels. These results suggest that decreased L-arginine availability may contribute to coronary arterial endothelial dysfunction in obese ZR. Thus, L-arginine supplementation may present a novel therapeutic method to deter coronary vascular endothelial dysfunction and cardiovascular complications in patients with obesity and type 2 diabetes.