Adenosine and lidocaine (AL) as a vasodilator in cardiac procedures and a storage solution for vascular banking

Arsyad, M. Aryadi (2018) Adenosine and lidocaine (AL) as a vasodilator in cardiac procedures and a storage solution for vascular banking. PhD thesis, James Cook University.

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Introduction: Cardiovascular disease is one of the most common causes of morbidity and mortality worldwide, and globally has contributed to more than 17 million deaths in a year. Coronary heart disease (CHD) alone is responsible for one in seven deaths in the US, and the mortality rate is expected to rise by 10% per year over the next 20 years. One of the invasive treatments for CHD is coronary artery bypass grafting (CABG), which aims to improve cardiac tissue perfusion by grafting another blood vessel to bypass the narrowed or blocked coronary artery. Currently the artery conduit is the standard choice for this procedure, however, the main concern for the use of artery conduits is that they have a high probability of inducing perioperative vasospasm. Therefore, it is crucial to maintain functionality of the conduit during harvest, pressure testing, storage and implantation.

The current strategy to prevent artery vasospasm involves a range of anti-spasmodic agents. Some of the most commonly used vasodilators in the surgical setting include Ca²⁺ antagonists (diltiazem, verapamil), nitrates (nitroglycerin, glyceryl trinitrate), and phosphodiesterase inhibitors (papaverine). However, the results remain unsatisfactory. Accordingly, the search for a vasorelaxation agent to reduce graft spasm remains an ongoing pursuit, which if successful may also be applicable to vascular surgery and neurosurgery. The aim of this thesis is to explore the use of adenosine and lidocaine combination as a potential vasodilator to improve arterial grafting using in vitro models.

Methods: In this thesis, vascular reactivity was assessed using two different in vitro methods: 1) Isometric force measurements for the isolated male rat aortic ring studies, and 2) Pressured myography for the isolated guinea pig mesenteric artery studies. Isometric force measurements of vasoreactivity were used as the basis for Chapters 3, 4 and 5; and for Chapter 6 after static cold storage. The pressure myography system was only used in Chapter 5.

Chapter 3 investigates the relaxation effect of adenosine as a single drug on rat aorta as well as its possible mechanisms of action. In this chapter, aortic rings were freshly harvested from adult male Sprague Dawley rats and equilibrated in an organ bath containing oxygenated, modified Krebs Henseleit (KH) solution (11 mM glucose, pH 7.4, 37°C). Isolated rings were pre-contracted sub-maximally with 0.3 μM norepinephrine (NE), and the effect of increasing concentrations of adenosine (1 to 1000 μM) was examined. The effect of antagonists on adenosine relaxation, such as Nᴳ-nitro-L-arginine methyl ester (L-NAME), indomethacin, 4-aminopyridine (4-AP), glibenclamide, 5-hydroxydecanoate (5-HD), ouabain, 8-(3-chlorostyryl) caffeine (CSC) and 8-[4-[4-(4-chlorobenzyl)piperazide-1-sulfonyl)phenyl]]-1-propylxanthine (PSB- 0788) were examined in intact and denuded aortic rings. Rings were dilated with 100 μM papaverine after each experiment to confirm viability.

In Chapter 4, lidocaine effects and mechanisms of action on rat aorta vasorelaxation were examined. Incremental concentrations of lidocaine (1 to 1000 μM) were administered and tested against 0.3 μM NE pre-contracted rat aorta. The effects of antagonists L-NAME, indomethacin, 4-AP, glibenclamide, 5-HD, ouabain, CSC and PSB-0788 were also examined against lidocaine relaxation. As in Chapter 3, rings were tested for viability after each experiment with maximally dilating 100 μM papaverine.

Chapter 5 focused on the effect of the combination of adenosine and lidocaine on rat aortic ring relaxation compared to each drug alone. Rings were pre-contracted submaximally with 0.3 μM norepinephrine, and the effects of increasing AL, A or L (up to 1.0 mM) were examined in intact and denuded rings. In this Chapter 5, the vasorelaxation effect of AL as a combination was further explored in the mesenteric artery of guinea pig. This study used the pressure myograph system to examine mesenteric conduit relaxation and the vascular dilatory response to adenosine, lidocaine and AL during luminal and abluminal administration. This methodology is often used to investigate small vessel function (diameter >60 μM) under near physiological conditions of pressure and flow by measuring vessel diameter and flow in time. Mesenteric artery segments were isolated from guinea pigs and mounted in an arteriograph containing KH solution and pressurized to 60 mmHg. Arteries were preconstricted with 10⁻⁸ M vasopressin and AL, A or L was administered luminally or abluminally. Diameters were measured using video-microscopy.

Chapter 6 explores the potential use of AL as an additive in a vessel preservation solution. In this chapter, thoracic aortic vessels were harvested from 300-350 g Sprague Dawley rats and transferred to a container with pre-cooled KH solution. Vessel segments were cleaned and cut in 3-mm length rings and stored at 4°C for six days in one of the following preservation solutions: 1) Krebs Henseleit (KH), 2) modified KH (low Ca²⁺/high Mg²⁺), 3) modified KH + adenosine-lidocaine (KH+AL), or 4) modified KH + AL and melatonin and insulin (KH+ALMI). After 6-day storage, physiological (contraction and relaxation) function of the preserved aortic rings was measured using an isometric force transducer. Contraction was induced by norepinephrine (NE; 0.3 μM) and potassium chloride (KCl; 60 mM). Vessel relaxation in response to acetylcholine (ACh; 10⁻⁶-10⁻³ M) and sodium-nitroprusside (SNP; 10-6- 10-3 M) was tested after preconstriction with 0.3 μM NE. At the end of each experiment, rings were maximally dilated with 100 μM papaverine to confirm viability of the vessels.

Results: Adenosine induced a dose-dependent, triphasic relaxation response, and the mechanical removal of the endothelium significantly decreased adenosine relaxation above 10 μM. Interestingly, endothelial removal significantly reduced the responsiveness (defined as % relaxation per μM adenosine) by two-thirds between 10 and 100 μM, but not in the lower (1-10 μM) or higher (>100 μM) ranges. In intact rings, L-NAME, but not indomethacin, significantly reduced relaxation, suggesting a role of nitric oxide (NO) but not prostacyclin in adenosine endothelium-dependent relaxation. Antagonists of voltage-dependent Kᵥ (4-AP), sarcolemmal K(ATP) (glibenclamide) and mitochondrial K(ATP) channels (5-HD) led to significant reductions in adenosine relaxation in both intact and denuded rings, with the Na⁺/K⁺-ATPase antagonist ouabain having little or no effect. Adenosine-induced relaxation appeared to involve the A₂ₐ receptor, but not the A₂(b) subtype. In contrast to adenosine, lidocaine relaxation in intact rings was biphasic between 1 to 10 μM (Phase 1) and 10 to 1000 μM (Phase 2). Mechanical removal of the endothelium resulted in further relaxation, and at lower concentrations ring sensitivity (% relaxation per μM lidocaine) significantly increased 3.5 times compared to intact rings. The relaxing factor(s) responsible for enhancing lidocaine relaxation did not appear to be NO- or prostacyclin-dependent, as L-NAME and indomethacin had little or no effect on intact ring relaxation. In denuded rings, lidocaine relaxation was completely abolished by Kᵥ channel inhibition and significantly reduced by antagonists of the MitoK(ATP) channel, and to a lesser extent the SarcK(ATP) channel. Curiously, A₂ₐ subtype receptor antagonism significantly inhibited lidocaine relaxation above 100 μM, but not the A₂(b) receptor. In combination, adenosine and lidocaine (AL) increased aortic relaxation from 21 to 100% (0.1-1.0 mM) and relaxation was endothelium-independent. Although adenosine alone was also a potent relaxant of aortic rings, unlike AL relaxation, it was partially endothelium-dependent.

Further investigation of AL effects on mesenteric artery showed that increasing luminal administration of AL in intact mesenteric artery segments produced a potent endothelium-independent dilation up to 90% (p<0.05). Adenosine dilation was endothelium-independent but not lidocaine, which produced 33% dilation only after endothelial removal. Extra-luminal AL and A led to 76% and 80% dilation in intact segments respectively, whereas L resulted in constriction (10-17%).

When exploring the potential use of AL as a preservation solution with 6-day cold storage in Chapter 6, it was found that AL addition in modified KH solution resulted in 100% recovery of NE contractile function in rat aorta, which was superior compared to KH solution alone (89% recovery). However, there was no further recovery in the KCl response over modified KH (76% recovery). A similar result was also shown with ALMI in modified KH, which led to 100% and 86% of contractile function recovery in response to NE and KCl, respectively. Furthermore, AL but not ALMI addition in modified KH significantly improved relaxation function compared to standard KH, with 93% recovery compared to 79% with modified KH alone after six days of storage. Maximal SNP relaxation following 6-day cold storage with either modified KH alone, modified KH with AL or with ALMI recovered 100%.

Conclusions: Adenosine is a potent vasodilator of aortic rings. Adenosine relaxation in NEprecontracted rat aortic rings was triphasic and endothelium-dependent above 10 μM, and relaxation involved endothelial nitric oxide (not prostanoids) and a complex interplay between smooth muscle A₂ₐ subtype and voltage-dependent Kᵥ, SarcK(ATP) and MitoK(ATP) channels. In contrast, lidocaine relaxation is not as potent as adenosine relaxation, but it appears to be significantly enhanced by endothelial removal, which did not appear to be NO- or prostacyclin-dependent. The unknown factor(s) responsible for enhanced relaxation was significantly reduced by Kᵥ channel inhibition, MitoK(ATP) channel inhibition, and A₂ₐ subtype inhibition indicating a potential role for crosstalk in lidocaine's vasoreactivity. When combined, AL can dilate aortic rings and mesenteric artery segments by up to 90% regardless of whether the endothelium is intact. This may have potential translational significance of AL to improve conduit protection in cardiac surgery, and other major surgeries where varying degrees of endothelial damage, vasoconstriction or vasospasm are known to occur. In addition, AL has a potential role as an adjuvant in preservation solutions since it improved vascular function after 6-day cold storage. AL addition in modified KH solution significantly improved NE-induced vascular contractility and ACh-induced relaxation compared to standard KH solution. This may indicate that AL improved endothelial preservation during storage, which was not achieved with standard preservation solution.

Item ID: 58873
Item Type: Thesis (PhD)
Keywords: adenosine, artery, CABG, compliance, endothelium, lidocaine, mesenteric, nitric oxide, rat aorta, redox stress, relaxation, vascular tone, vasodilation, vasospasm ventricular-arterial coupling
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Copyright Information: Copyright © 2018 M. Aryadi Arsyad.
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Publications arising from this thesis are available from the Related URLs field. The publications are:

Chapter 3: Arsyad, Aryadi, and Dobson, Geoffrey P. (2016) Adenosine relaxation in isolated rat aortic rings and possible roles of smooth muscle Kv channels, KATP channels and A2a receptors. BMC Pharmacology and Toxicology, 17 (23). pp. 1-11.

Chapter 3: Dobson, Geoffrey P., Arsyad, Aryadi, and Letson, Hayley (2017) The adenosine hypothesis revisited: modulation of coupling between myocardial perfusion and arterial compliance. Frontiers in Physiology, 8. 824.

Chapter 4: Arsyad, Aryadi, and Dobson, Geoffrey P. (2016) Lidocaine relaxation in isolated rat aortic rings is enhanced by endothelial removal: possible role of Kv, KATP channels and A2a receptor crosstalk. BMC Anesthesiology, 16 (121). pp. 1-11.

Chapter 5: Arsyad, Aryadi, Sokoya, Elke, and Dobson, Geoffrey P. (2018) Adenosine and lidocaine (AL) combination dilates intimally damaged rat thoracic aortic rings and guinea pig mesenteric arteries: possible significance to cardiac surgery. American Journal of Translational Research, 10 (6). pp. 1841-1851.

Date Deposited: 09 Jul 2019 04:52
FoR Codes: 11 MEDICAL AND HEALTH SCIENCES > 1102 Cardiovascular Medicine and Haematology > 110201 Cardiology (incl Cardiovascular Diseases) @ 60%
11 MEDICAL AND HEALTH SCIENCES > 1109 Neurosciences > 110903 Central Nervous System @ 40%
SEO Codes: 92 HEALTH > 9201 Clinical Health (Organs, Diseases and Abnormal Conditions) > 920103 Cardiovascular System and Diseases @ 100%
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