Endothelin-Induced Sarcoplasmic Reticulum Calcium Depletion Waves in Vascular Smooth Muscle Cells

Agonist-stimulated waves of elevated cytoplasmic Ca2+ concentration ([Ca2+]i ) regulate blood vessel tone and vasomotion in vascular smooth muscle. Previous studies employing cytoplasmic Ca2+ indicators revealed that these Ca2+ waves were generated by a combination of inositol 1,4,5-trisphosphate (IP3) and Ca2+ induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR); although, some of the mechanistic details remain uncertain. However, these findings were derived indirectly from observing agonist-induced [Ca2+]i fluctuations in the cytoplasm.
Here, for the first time, we have recorded Endothelin-1 (ET-1) induced waves of Ca2+ depletion from the SR lumen in vascular smooth muscle cells (VSMCs) using a calsequestrin-targeted Ca2+ indicator. Our findings show that these waves: (1) are due to regenerative CICR by the receptors for IP3 (IP3R), (2) have a marked latency period, (3) are characterized by a transient increase in SR Ca2+ ([Ca2+]SR ) both at the point of origin and at the wave front, (4) proceed with diminishing velocity, and (5) are arrested by the nuclear envelope. Our quantitative model indicates that the gradual decrease in the velocity of the SR depletion wave, in the absence of external Ca2+, results from continuity of the SR luminal network

Influence of nanosilver on endothelial function and vascular reactivity of isolated rabbit carotid artery

Summary: There is paucity of information on the effects and mechanism of action of Nanosilver on vascular tone and endothelial function in spite of the upsurge in nanotechnology application in biomedicine. The present study determined the effect of Nanosilver on vascular reactivity and endothelial function on isolated rabbit carotid artery in standard laboratory 20 mL organ bath procedures containing physiological salt solution (PSS) bubbled with 95% O2, 5% CO2. Isometric contractions were recorded electronically with a 4-channel Grass Polygraph and maintained at 37oC and pH7.4. Cumulative dose response tests to α-receptor agonist phenylephrine (PE) was examined separately, in normal PSS (control) and following 20 minutes exposure to varying concentrations of Nanosilver solution [(NAgs) (1.25 and 2.50)] μg/mL in endothelium intact (+E) (control) and endothelium denuded (-E) rings. Contractile responses were analysed with reference to maximal contractions induced by 8 x 10-2 M K+ in normal PSS. In another experiment, arterial rings were precontracted with EC70 M PE, high and /or low (8, 2 x 10-2) M K+PSS. At stable contractions, cumulative relaxation responses to NAgs was studied. Relaxation responses were analysed with reference to maximal contraction induced by EC70 M PE and/or K+ depolarization in normal PSS. Following 20 minutes exposure to NAgs, dose relaxation response to acetylcholine (ACh) was also examined in normal PSS (control), and pre-incubated L-NAME (NO synthase inhibitor) and indomethacin (cyclooxygenase inhibitor) precontracted arterial rings to further determine mechanisms of action. Data were presented as Means ± SEM. Graphs and statistical analysis were done using GraphPad prism version 7.03 and Student t-test. P-values (P< 0.05) were considered statistically significant. The results showed that nanosilver decreased maximum contraction (Emax) and induced attenuated contractile and relaxation responses concentration-dependently in +E and –E carotid arterial rings. Also, Nanosilver-induced relaxation in α- receptor mediated contraction is endothelium-dependent and showed a biphasic dose-dependent response. In conclusion, Nanosilver causes attenuation in carotid arterial smooth muscle reactivity with a biphasic dose-dependent relaxant effect and multiple endothelium-dependent pathways mode of action.Keywords: Nanosilver, Vascular reactivity, Carotid arterial rings, Vascular endotheliumNiger. J. Physiol. Sci. 33(December 2018) 139-14

Bioabsorbable metal zinc differentially affects mitochondria in vascular endothelial and smooth muscle cells

Zinc is an essential trace element having various structural, catalytic and regulatory interactions with an estimated 3000 proteins. Zinc has drawn recent attention for its use, both as pure metal and alloyed, in arterial stents due to its biodegradability, biocompatibility, and low corrosion rates. Previous studies have demonstrated that zinc metal implants prevent the development of neointimal hyperplasia, which is a common cause of restenosis following coronary intervention. This suppression appears to be smooth muscle cell-specific, as reendothelization of the neointima is not inhibited. To better understand the basis of zinc\u27s differential effects on rat aortic smooth muscle (RASMC) versus endothelial (RAENDO) cells, we conducted a transcriptomic analysis of both cell types following one-week continuous treatment with 5 µM or 50 µM zinc. This analysis indicated that genes whose protein products regulate mitochondrial functions, including oxidative phosphorylation and fusion/fission, are differentially affected by zinc in the two cell types. To better understand this, we performed Seahorse metabolic flux assays and quantitative imaging of mitochondrial networks in both cell types. Zinc treatment differently affected energy metabolism and mitochondrial structure/function in the two cell types. For example, both basal and maximal oxygen consumption rates were increased by zinc in RASMC but not in RAENDO. Zinc treatment increased apparent mitochondrial fusion in RASMC cells but increased mitochondrial fission in RAENDO cells. These results provide some insight into the mechanisms by which zinc treatment differently affects the two cell types and this information is important for understanding the role of zinc treatment in vascular cells and improving its use in biodegradable metal implants

Pharmacological agents that distinguish between P2X receptor subtypes.

The activity of novel pharmacological agents at recombinant P2X receptors was studied to find agents that distinguish between P2X receptor subtypes, particularly P2X1 and P2X3. Adenine nucleotide derivatives and diadenosine polyphosphates (ApnA, n = 2-6) were investigated as P2X receptor agonists. PAPET and HT-AMP were agonists, to varying degrees, at P2X1-4 receptors. PAPET displayed higher affinity but lower efficacy than ATP at P2X1 and P2X3 receptors. HT-AMP showed higher affinity than ATP at P2X3 receptors yet acted as a partial agonist at P2X1-4 receptors. Diadenosine polyphosphates also showed selectivity in their actions at P2X1-4 receptors. Ap2A was inactive and Ap3-6A showed varying affinities and efficacies as agonists at P2X1-4. Ap3A was most effective at distinguishing between P2X1 and P2X3 receptors with over 100 fold difference between their respective EC50 values A series of PPADS derivatives, involving chemical manipulation of the phenylazo moiety and/or the pyridoxal phosphate moiety, showed nanomolar activity at P2X1 and P2X3 receptors with variable degrees of selectivity between these receptor subtypes. The most potent compounds were studied in detail and shown to be nonsurmountable antagonists. A comparison of like data for recombinant P2X1 receptors and native P2X1-like receptors in vas deferens revealed a number of pharmacological anomalies. Co-expression of P2X1 and P2X2 revealed a novel pH-sensitive phenotype although this heteromeric receptor is unlikely to account for the difference between rP2X1 and the native P2X subtype(s) in this tissue. A step forward has been made in the search of pharmacological agents that distinguish between P2X1 and P2X3 receptors. Antagonist-resistant ATP responses in the vas deferens lend weight for other contraction-mediating P2 receptors in this preparation. Greater diversity of purinergic signalling was revealed through co-expression of P2X receptors and underlines the need for further novel pharmacological tools

Lidocaine relaxation in isolated rat aortic rings is enhanced by endothelial removal: possible role of Kv, KATP channels and A2a receptor crosstalk

Background: Lidocaine is an approved local anesthetic and Class 1B antiarrhythmic with a number of ancillary properties. Our aim was to investigate lidocaine's vasoreactivity properties in intact versus denuded rat thoracic aortic rings, and the effect of inhibitors of nitric oxide (NO), prostenoids, voltage-dependent Kv and KATP channels, membrane Na+/K+ pump, and A2a and A2b receptors. Methods: Aortic rings were harvested from adult male Sprague Dawley rats and equilibrated in an organ bath containing oxygenated, modified Krebs-Henseleit solution, pH 7.4, 37 °C. The rings were pre-contracted sub-maximally with 0.3 μM norepinephrine (NE), and the effect of increasing lidocaine concentrations was examined. Rings were tested for viability after each experiment with maximally dilating 100 μM papaverine. The drugs 4-aminopyridine (4-AP), glibenclamide, 5-hydroxydecanoate, ouabain, 8-(3-chlorostyryl) caffeine and PSB-0788 were examined. Results: All drugs tested had no significant effect on basal tension. Lidocaine relaxation in intact rings was biphasic between 1 and 10 μM (Phase 1) and 10 and 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 ring 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 MitoKATP channel, and to a lesser extent the SarcKATP channel. Curiously, A2a subtype receptor antagonism significantly inhibited lidocaine relaxation above 100 μM, but not the A2b receptor

Mitogenic Signalling by the Endothelin Receptor in Rat-1 Fibroblasts

Endothelin-1 (ET-1) was found to be a complete mitogen in Rat-1 fibroblasts and therefore the signalling pathways which might mediate its action were investigated. ET-1 stimulated both sustained phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) and phosphatidylcholine (PtdCho) hydrolysis. The rank order of potency for both pathways stimulated by a range of ET isopeptides was the same for both responses (ET-1 ~ ET-2 > ET-3) suggesting that the same receptor controls both signalling pathways. PtdCho hydrolysis occurred kinetically downstream of Ptdlns(4,5)p2 hydrolysis by a phospholipase D-catalysed mechanism apparently involving both protein kinase C-dependent and -independent means of activation

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

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

The Effects of Drugs on Conduction and Transmission in Autonomically-Innervated Smooth Muscle

1. The prejunctional actions of a number of Ca 2+ channel antagonists were investigated. Their effects were assessed on both nerve action potential (AP) conduction and neuromuscular transmission in the vas deferens and internal anal sphincter (i.a.s.) of the guinea-pig and in the rat anococcygeus. Nerve APs were recorded extracellularly with a suction electrode and transmitter release measured by intracellular electrical recording of smooth muscle junction potentials and analysis of the overflow of radiolabelled transmitter

Bacillus anthracis edema factor substrate specificity: evidence for new modes of action

Since the isolation of Bacillus anthracis exotoxins in the 1960s, the detrimental activity of edema factor (EF) was considered as adenylyl cyclase activity only. Yet the catalytic site of EF was recently shown to accomplish cyclization of cytidine 5'-triphosphate, uridine 5'-triphosphate and inosine 5'-triphosphate, in addition to adenosine 5'-triphosphate. This review discusses the broad EF substrate specificity and possible implications of intracellular accumulation of cyclic cytidine 3':5'-monophosphate, cyclic uridine 3':5'-monophosphate and cyclic inosine 3':5'-monophosphate on cellular functions vital for host defense. In particular, cAMP-independent mechanisms of action of EF on host cell signaling via protein kinase A, protein kinase G, phosphodiesterases and CNG channels are discussed