Modified Krebs-Henseleit Solution Research Articles

O100 The effect of acidosis on contractility induced in porcine splanchnic arteries by clonidine or noradrenaline

Abstract Introduction Clonidine, through its agonist action on central α2-adrenoceptors, is increasingly being used to induce sedation in critically ill patients with persistent acidosis and impaired renal function. Since it is also known to exert cardiovascular effects via peripheral α2-adrenoceptors, we compared its vasoconstrictor activity in porcine isolated arteries at different pH, to that produced by noradrenaline. Methods Isometric tension recordings of porcine isolated mesenteric, splenic and renal arteries (2mm internal diameter) were conducted in modified Krebs-Henseleit solution containing 105mM chloride ions maintained at pH 7.2 and 7.4 (modification of bicarbonate and chloride ions with gluconate ions). The potency of clonidine and noradrenaline was assessed by the pD2 (logarithm of concentration causing 50% of maximum response). Results Clonidine elicited concentration-dependent contractions in the renal artery (pD2 - 7.25±0.09 and 7.10±0.10, n=9) and splenic artery (pD2 - 6.81±0.09 and 7.02±0.06, n=8) that were not altered by a reduction in pH from 7.4 to 7.2 (p >0.05 paired Student's t-test). Clonidine failed to elicit consistent responses in the mesenteric artery (< 10% 60mM KCl). In contrast, the maximum response and potency to noradrenaline in the mesenteric artery (226.6±24.6% and pD2 5.43±0.11, n=6) was significantly reduced (p <0.05) by pH 7.2 (174.4±13.3% and pD2 5.13±0.11, n=6). Responses to noradrenaline in the renal and splenic artery were not affected by changes in pH. Conclusion Acidosis impairs the peripheral vascular actions of the non-selective α-adrenoceptor agonist noradrenaline more than those elicited by the selective α2-adrenoceptor agonist clonidine.

The Effects of Curcumin on Mechanical Functions and Cardiac Contractility in Isolated Rat Hearts.

Curcumin is a bioactive compound that has well-known pharmacological activities. Numerous studies have shown that curcumin provides potential cardiovascular benefits through a variety of mechanisms.The present study aims to discuss different concentrations of curcumin's impact on mechanical functions and cardiac contractility in isolated perfused rat hearts. The hearts were isolated under sodium thiopental (50 mg/kg) anesthesia and perfused with a modified Krebs-Henseleit solution (mK-Hs). After stabilization, curcumin was applied in concentrations of 0.1, 1, and 10 µM. In isolated rat hearts, indexes of + dP / dt max, LVDP, MAP, and LVEDP were evaluated for cardiac contractility and ventricular function. All curcumin concentrations reduced +dP/dtmax and LVDP. Ten µM curcumin also significantly decreased heart rate. Curcumin (1 and 10 µM) increased LVEDP and reduced MAP amplitude with a concomitant increase in MAP duration. Curcumin at all concentrations did not affect dMAPdtmax and dMAPdtmin. Our results might suggest that curcumin at higher concentrations (≥ 1 µM) increases LVEDP with a negative chronotropic effect and decreases MAP amplitude with an increase in MAP duration. There is sufficient evidence from this study that Curcumin possesses an adverse inotropic action. Different disease models should support the pathophysiological role of Curcumin on cardiac contraction.

Proof of concept study for a closed ex vivo limb perfusion system for 24-hour subnormothermic preservation using acellular perfusate.

The two approaches to vascularized tissue machine perfusion use either the open (nonpressurized) or closed (pressurized) perfusion system. Most studies describing isolated limb perfusion preservation rely on open perfusion systems and report tissue edema exceeding 40% after 12 to 14 hours of preservation. A variant of machine perfusion places the limb and perfusate into a reservoir closed to atmosphere. It is hypothesized that the reservoir pressure, acting as a transmural pressure, has the advantage of reducing edema formation by counteracting the hydrostatic pressure gradient from the perfusion pressure. This proof-of-concept study aim was to demonstrate feasibility of the Universal Limb Stasis System for Extended Storage (ULiSSES) device (closed, vertical perfusion system) to preserve forelimbs of Sus scrofa swine for 24 hours of subnormothermic perfusion compared with an open, horizontal perfusion system. The ULiSSES is a compact, practical device that applies pulsatile, pressurized perfusion through the novel use of a diaphragm pump powered by compressed oxygen. Forelimbs from swine were preserved in ULiSSES device (closed perfusion system) (n = 9) and in an open perfusion system (n = 4) using subnormothermic modified Krebs-Henseleit solution. Physiological parameters were measured at the start and every 3 hours for 24 hours. Limbs were weighed before and after perfusion to compare weight gain. Edema and cellular integrity were evaluated using histopathology pre and post perfusion. Closed perfusion system showed superiority compared with the open perfusion system in terms of oxygen consumption, reduction in vascular resistance, and overall tissue integrity. The closed perfusion system demonstrated a 21% reduction in weight gain compared with the open perfusion system and significantly reduced intracellular edema. The ULiSSES closed, pressurized perfusion technology has translatable military applications with the potential to preserve porcine limbs for 24 hours with improved results compared with an open perfusion system.

Phosphodiesterase-5 inhibitor sildenafil attenuates kidney injury induced by Bothrops alternatus snake venom

Acute kidney injury pathogenesis in envenoming by snakes is multifactorial and involves immunologic reactions, hemodynamic disturbances, and direct nephrotoxicity. Sildenafil (SFC), a phosphodiesterase 5 inhibitor, has been reported to protect against pathological kidney changes. ObjectiveThis study aimed to investigate the protective effect of sildenafil against Bothrops alternatus snake venom (BaV)-induced nephrotoxicity. MethodsKidneys from Wistar rats (n = 6, weighing 260–300 g) were isolated and divided into four groups: (1) perfused with a modified Krebs-Henseleit solution (MKHS) containing 6 g% of bovine serum albumin; (2) administered 3 μg/mL SFC; (3) perfused with 3 μg/mL BaV; and (4) administered SFC + BaV, both at 3 μg/mL. Subsequently, the perfusion pressure (PP), renal vascular resistance (RVR), urinary flow (UF), glomerular filtration rate (GFR), and percentage of electrolyte tubular sodium and chloride transport (%TNa+, %TCl−, respectively) were evaluated. The cyclic guanosine monophosphate (cGMP) levels were analyzed in the perfusate, and the kidneys were removed to perform oxidative stress and histopathological analyses. ResultsAll renal parameters evaluated were reduced with BaV. In the SFC + BaV group, SFC restored PP to normal values and promoted a significant increase in %TNa+ and %TCl−. cGMP levels were increased in the SFC + BaV group. The oxidative stress biomarkers, malondialdehyde (MDA) and glutathione (GSH), were reduced by BaV. In the SFC + BaV group, a decrease in MDA without an increase in GSH was observed. These findings were confirmed by histological analysis, which showed improvement mainly in tubulis. ConclusionOur data suggest the involvement of phosphodiesterase-5 and cGMP in BaV-induced nephrotoxicity since its effects were attenuated by the administration of SFC.

Vasorelaxant Effects of Coriandrum SativumL. Extract on Rat Isolated Aorta

Aim: Coriander(Coriandrum sativum L.) is an annual plant belonging to the family Umbelliferae, which distributed in Central and Western Europe, India, Bangladesh, Thailand, China, and other Asian regions. Besides being edible, coriander is an important traditional medicine in India and China, and is used to treat circulatory disorders, such as respiratory, urinary tract, and skin diseases. In this study, we investigated the vasorelaxant effects of extracts from coriander and further studied to clarify their action mechanisms. Method: The aerial part of coriander, which analyzed the rutin content as its quality evaluand using a chromatographic method (HPLC), was cut and extracted with ethyl acetate or hot water. The extracts were concentrated under the reduced pressure. Vasorelaxant effects of these extracts were assessed on rat isolated aorta. The aorta was placed in a well-oxygenated bath of modified Krebs-Henseleit solution and the mechanical tension was measured isometrically. Results: These extracts showed vasorelaxant effects on aorta precontracted with 3×10-7 M norepinephrine (NE). The ethyl acetate extract showed biphasic vasorelaxation (fast and slowly developing relaxations) on isolated rat aortic rings with endothelium. Fast relaxation disappeared in deendothelialized or pre-administration of 10-4 M L-NG-monomethylarginine. Furthermore, the hot water extract showed only slowly developing relaxations independent in endothelium. After treatment with the hot water extract, NE-induced phasic vasoconstriction was not inhibited. While the hot water extract inhibited vasoconstrictions induced by a high concentration (60 mM) of K+ and also showed inhibitory effect on NE-induced vasoconstriction in the presence of nicardipine. Conclusion: These results suggest that the vasorelaxant effect of ethyl acetate extract of coriander on NE-induced vasoconstriction may be attributed to Nitric Oxide (NO) releasing dependent on endothelium. And the hot water extract of coriander showed vasorelaxant activities attributed to blocking of Ca2+ influx via voltage-depended Ca2+ channels (VDCs) and receptor-operated Ca2+ channels (ROCs), but not competing for the adrenergic receptor. Keywords: Coriandrum sativum; quality evaluation; vasorelaxant effects; aorta; endothelium; Ca2+ channel

Aprotinin improves myocardial recovery after ischemia and reperfusion. Effects of the drug on isolated rat hearts.

The effects of aprotinin, a protease inhibitor, on the ischemic and nonischemic isolated rat heart was investigated with the use of the modified Langendorff model. During phase I of the study, hearts were perfused with either low-dose aprotinin (10(5) KIU/L), high-dose aprotinin (10(6) KIU/L), or normal saline solution added to modified Krebs-Henseleit solution. No statistically significant differences in contraction amplitude, contractility, coronary flow, and wet/dry heart weight ratio were observed among the three groups of hearts. In phase II, hearts were exposed to a 40-minute period of global ischemia at 31 degrees C. Ischemic arrest was induced by warm cardioplegia. Before ischemia and during cardioplegia, hearts were perfused with either aprotinin 10(6) KIU/L (n = 10) or normal saline solution (n = 10) for 30 minutes. On reperfusion, recovery of hearts treated with aprotinin was significantly better than that of control hearts, as reflected by better contractility (analysis of variance, p = 0.011), higher coronary flow (p < 0.025), and lower creatine kinase levels (p < 0.05). No statistically significant differences in contraction amplitude were observed between the two groups. When the effect of ischemia within each group of hearts was analyzed, the preserving effect of aprotinin was even more pronounced. In the control group, ischemia caused a decrease in contractility (p < 0.025) and a decrease in oxygen consumption (p = 0.006); by contrast, in the aprotinin group the preischemic values were maintained. Accordingly, we conclude that aprotinin at concentrations up to 10(6) KIU/L has no deleterious effect on normally perfused hearts and has a significant protective effect on the ischemic heart when used in high doses in the preischemic period.

Progesterone pretreatment reduces the incidence of drug-induced torsades de pointes in atrioventricular node-ablated isolated perfused rabbit hearts.

Higher progesterone concentrations are protective against drug-induced prolongation of ventricular repolarization. We tested the hypothesis that pretreatment with progesterone reduces the incidence of drug-induced torsades de pointes (TdP). Female New Zealand white rabbits (2.5-3.2 kg) underwent ovariectomy and were randomized to undergo implantation with subcutaneous 21-day sustained release pellets containing progesterone 50 mg (n = 22) or placebo (n = 23). After 20 days, hearts were excised, mounted, and perfused with modified Krebs-Henseleit solution. The atrioventricular (AV) node was destroyed manually. Following a 15-minute equilibration period, hearts were perfused with dofetilide 100 nM for 30 minutes, during which the electrocardiogram was recorded continuously. Incidences of spontaneous TdP, other ventricular arrhythmias and mean QTc intervals were compared. Median serum progesterone concentrations were higher in progesterone vs placebo-treated rabbits (3.8 [range, 2.8-5.1] vs 0.7 [0.4-1.7] ng/mL, P < 0.0001). Median serum estradiol concentrations were similar (58 [22-72] vs 53 [34-62] pg/mL), P = 0.79). The incidence of TdP was lower in hearts from progesterone-treated rabbits (27% vs 61%, P = 0.049). The incidences of bigeminy (36% vs 74%, P = 0.03) and trigeminy (18% vs 57%, P = 0.01) were also lower in hearts from progesterone-treated rabbits. There was no significant difference between groups in incidence of couplets (59% vs 74%, P = 0.54) or monomorphic ventricular tachycardia (14% vs 30%, P = 0.28). Maximum QT c interval and short-term beat-to-beat QT interval variability during dofetilide perfusion were significantly shorter in hearts from progesterone-treated rabbits. Pretreatment with progesterone reduces the incidence of drug-induced TdP, bigeminy, and trigeminy in isolated perfused AV node-ablated rabbit hearts.

Renal Effects Induced by Two Phospholipase A2 (Asp‐49 and Lys‐49) Isolated from Bothropoides pauloensis Snake Venom

Acute renal failure (ARF) wich is one of the most serious complications of Bothrops snakebites, is one of the main causes of death and consequences for victims. However, its pathogenesis remains unclear. Considering that isolated toxins are relevant tools for understanding the actions of the whole venom, we studied properties of phospholipases A2 (Asp‐49 and Lys‐49) isolated from Bothrops pauloensis (from State of São Paulo, Southeast region of Brazil), on isolated perfused kidney of rat. The control group perfused with modified Krebs–Henseleit solution alone. The PLA2s (3μg/mL) caused significant effects in isolated rat kidney, Asp‐49 reducing perfusion pressure(PP), the Renal Vascular Resistence (RVR), the Urinary Flow (UF), the Glomerular Filtration Rate (GFR) and the percentage of tubular sodium (%TNa+), chloride (%TCl−) and potassium (%TK+). The Lys‐49 increased PP and UF and decreased the RFG and the %TNa+, %TK+ and %TCl‐. Oxidative stress in kidney tissue was evaluated by TBARS (thiobarbituric acid reactive substances), Nitrite and reduced Glutathione (GSH) level measurement. A significant increase was observed in TBARS (Lys‐49) and Nitrito (Asp‐49 and Lys‐49) and a decrease in GSH levels (Asp‐49) and increase (Lys‐49). The levels of inflammatory cytokines were measured. We analized the inflammatory cytokines, pro‐inflammatory interleukin 1 (IL‐1), tumor necrosis factor‐α (TNF‐α) and anti‐inflammatory interleukin 10 (IL‐10). The results showed high levels of both cytokines (by Lys‐49) and incresed TNF‐α and IL‐10 by Asp‐49. Increased TBARS, Nitrito level and decreased of GSH activity might be implicated in the presence of oxidative stress in patients with snake bite envenomation. These findings demonstrated that PLA2s caused nephrotoxicity in isolated kidney. The characterization of the effects in the isolated kidney gives strong evidences that the acute renal failure induced by these PLA2s is a result of the direct nephrotoxicity which may involve oxidative and inflammatory mechanisms.Support or Funding InformationCAPES, CNPq, UFC.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of omentin on cardiovascular functions and gene expressions in isolated rat hearts.

Objective:Omentin is a recently identified novel adipocytokine mainly expressed in the epicardial adipose tissue. Although it has favorable effects on cardiovascular disease, the impact of omentin on the hearts is still an understudied issue. The aim of the present study was to investigate the possible effects of omentin on isolated rat heart.Methods:Using the Langendorff method, 28 adult male Sprague–Dawley rat hearts were isolated and perfused with modified Krebs–Henseleit solution (mK–Hs). Concentrations of 100, 200, and 400 ng/mL omentin were given to the hearts for 30 min. The control group (n=7) was perfused with mK–Hs alone. Gene expressions in the left ventricle tissue were determined by real-time polymerase chain reaction. Left ventricular cyclic adenosine monophosphate and cyclic guanosine monophosphate (cGMP) concentrations were determined by using enzyme-linked immunosorbent assay.Results:All concentrations of omentin significantly decreased left ventricular developed pressure and maximal rate of pressure development that are the indexes of cardiac contractility. At the same time, omentin decreased both phosphoinositide 3-kinase γ (PI3Kγ) and sarcolemmal L-type Ca2+ channel (CaV1.2) mRNA levels. Moreover, this peptide at concentrations of 200 and 400 ng/mL increased endothelial nitric oxide synthase (eNOS) mRNA. Furthermore, concentrations of 200 and 400 ng/mL omentin increased the amount of cGMP.Conclusion:We conclude that acute omentin treatment decreases cardiac contractility. Elevated eNOS mRNA and cGMP levels with reduced CaV1.2 mRNA are likely to lead to negative inotropy.

The impact of apelin-36 on isolated rat hearts as a member of apelin family.

Apelinis an endogenousadipocytokine thatplays an important role in the regulation of cardiovascular function. Apelin-36 is a member of apelin family. However cardiac reports related to apelin-36 are very rare. The purpose of this study is to investigate the impact of apelin-36 onhemodynamic variables of the isolated rat hearts. Twenty-eight ratswere equally divided into four groups: control, apelin-36 at the following concentrations: 1 nM, 10 nM and 100 nM. Theisolatedhearts were perfused with modified Krebs-Henseleit solution (mK-Hs) by using theLangendorffmethod. Cardiac parameters, including left ventricular developed pressure (LVDP),maximal rate of pressure development (+dP/dtmax), heartrate (HR) and coronary flow (CF) were measured. Gene expressions relevant to cardiomyocyte contractility were determined by real-time PCR. 10 and 100 nM doses of apelin-36 perfusion led topositive inotropy with an increase of LVDP and +dP/dtmax, which are the indexes of cardiac contractility. Furthermore both doses of apelin-36 increased endothelial nitric oxide synthase (eNOS), sarco/endoplasmic reticulum Ca2+-ATPase (Serca2a) andβ2-Adrenergic receptors (β2-AR) mRNA. These results showed that apelin-36 had a positive inotropic effect on the isolated rat heart and can induceeNOS,Serca2aandβ2-ARgenes activation that enhancescontractilityof theheart (Tab. 1, Fig. 2, Ref. 23).

40 Days Survival after Orthotopic Cardiac Xenotransplantation of Multi-Transgenic Pig Hearts in a Pig-to-Baboon Model with CD40mAb or CD40L Costimulation Blockade and Xenograft Preservation using “Steens” Cold Blood Cardioplegia Perfusion

Introduction: We used a non-toxic immunosuppression based on CD40/CD40L co-stimulation blockade in group G1 and additionally to improve primary xenograft function in a group G2 a new non-ischemic myocardial preservation technique to achieve a constant preclinical long-term survival in a life-supporting cardiac xenotransplantation model (pig-to-baboon). Methods: According to the technique of Lower and Shumway 9 orthotopic (OHXTx) heart transplantations were performed in baboons with genetically-modified GalKO/hCD46/hTM transgenic pig hearts. The immunosuppression (IS) consisted of ATG, rituximab, MMF, tapered down cortisone and CD40 antibody or PASylated Fab-CD40L. Because a “perioperative cardiac xenograft dysfunction” (PCXD) played a critical role, cardioplegia with crystalloid solution (Bretschneider solution, 50 ml/kg; G1: n = 5) was replaced in the last 4 cases of G2 by Steen´s “non-ischemic preservation technique”: This 8°C cold myocardial perfusion solution consists of a modified Krebs-Henseleit solution with albumin and 10% erythrocytes, hormones and vasoactive agents (Steen et al, 2016*). Under pressure, flow and temperature control heart is constantly perfused during explantation and storage time, intermittently during implantation, using an independent portable heart-lung-machine. Results: Ischemic time ranged from 112–128 min. Survival in G1 with Bretschneider solution were 3, 1, 30, 1 and 1 day(s). Using non-ischemic preservation technique in G2 PCXD was not observed and the recipients survived 18, 1**, 27 and 40 days. Baboons mostly died of respiratory problems, renal and hepatic failure, one was sacrificed due to a major p.o. neurological deficit**, another died after hematothorax, but no hyperacute or delayed xenograft rejection was found. A problem after 3 weeks was a donor organ (over)growth. All long-term surviving baboons (especially in G2) were in good general conditions. Conclusion: With conventional hypothermic, ischemic heart preservation techniques, perioperative cardiac xenograft dysfunction plays a detrimental role after orthotopic xHTx. This was prevented with the Steen’s “non-ischemic preservation technique, an important step on the way to a long-term survival of 2–3 months as an important prerequisite for cardiac xenotransplantation in a clinical use.

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

BackgroundLidocaine 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.MethodsAortic 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.ResultsAll 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.ConclusionsWe show that lidocaine relaxation in rat thoracic aorta was biphasic and 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 inhibition, 5-HD inhibition, and A2a subtype inhibition indicating a potential role for crosstalk in lidocaine’s vasoreactivity.

The Effects of Beta Amyloid Peptide 1-42 on Isolated Rat Hearts and Ileum Smooth Muscle

Neurotoxic beta amyloid peptides (βAPs) are involved in the pathogenesis of Alzheimer disease. βAP_1-42 may also play a role in the regulation of cardiovascular functions. Therefore, we investigated the possible effects of βAP_1-42 on isolated rat heart and ileum. The hearts were perfused with modified Krebs-Henseleit solution. Left ventricular developed pressure (LVDP), maximal rate of pressure development of left ventricle (+dP/dt_max), heart rate, coronary flow, monophasic action potential amplitude (MAPamp), MAP duration at 90% repolarization (MAP₉₀) and contractions of ileum were measured. One, 10 and 100 nmol/l doses of βAP_1-42 significantly decreased LVDP, +dP/dt_max and heart rate. The dose of 1 nmol/l did not change coronary flow, but 10 and 100 nmol/l doses significantly reduced it. All doses of βAP_1-42 did not alter MAPamp, but increased MAP₉₀. βAP_1-42 (1, 10, 100, 1,000 nmol/l) also did not influence ileum contractions. We suggest that βAP_1-42 produces negative inotropic and negative chronotropic effects with an increase in MAP duration. Furthermore, βAP_1-42 at high doses decreases coronary flow.

(-)-Epigallocatechin-3-O-gallate (EGCG) attenuates the hemodynamics stimulated by caffeine through decrease of catecholamines release.

A human study of the effects on hemodynamics of caffeine and epigallocatechin-3-O-gallate (EGCG) was performed. Caffeine tablets (200mg) were orally administered to healthy males aged between 25 and 35years 30min after oral administration of EGCG tablets (100 and 200mg). The increase in BP induced by caffeine was inhibited when co-administrated with EGCG. We found that caffeine slightly decreased heart rate (HR) in the volunteers. Although EGCG enhanced HR reduction, the effect was not significant. In addition, caffeine increased blood catecholamine levels, but EGCG inhibited the increase in noradrenaline, adrenaline and dopamine levels induced by caffeine. Whether EGCG decreases the elevated HR and systolic perfusion pressure, and ventricular contractility induced by adrenergic agonists in the isolated rat heart was investigated. The modified Krebs-Henseleit solution was perfused through a Langendorff apparatus to the isolated hearts of rats. HR, systolic perfusion pressure, and developed maximal rates of contraction (+dP/dtmax) and relaxation (-dP/dtmax) were increased by epinephrine (EP) and isoproterenol (IP). In contrast, EGCG decreased the elevated HR, systolic perfusion pressure, and left ventricular±dp/dtmax induced by EP and/or IP. In conclusion, EGCG could attenuate the hemodynamics stimulated by caffeine through decreasing catecholamine release.

Adenosine relaxation in isolated rat aortic rings and possible roles of smooth muscle Kv channels, KATP channels and A2a receptors.

BackgroundAn area of ongoing controversy is the role adenosine to regulate vascular tone in conduit vessels that regulate compliance, and the role of nitric oxide (NO), potassium channels and receptor subtypes involved. The aim of our study was to investigate adenosine relaxation in rat thoracic aortic rings, and the effect of inhibitors of NO, prostanoids, Kv, KATP channels, and A2a and A2b receptors.MethodsAortic rings were freshly harvested from adult male Sprague Dawley rats and equilibrated in an organ bath containing oxygenated, modified Krebs-Henseleit 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) were examined. The drugs L-NAME, indomethacin, 4-aminopyridine (4-AP), glibenclamide, 5-hydroxydecanoate, ouabain, 8-(3-chlorostyryl) caffeine and PSB-0788 were examined in intact and denuded rings. Rings were tested for viability after each experiment.ResultsAdenosine induced a dose-dependent, triphasic relaxation response, and the mechanical removal of the endothelium significantly deceased adenosine relaxation above 10 μM. Interestingly, endothelial removal significantly decreased 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 significantly reduced relaxation, but not indomethacin. Antagonists of voltage-dependent Kv (4-AP), sarcolemma KATP (glibenclamide) and mitochondrial KATP channels (5-HD) led to significant reductions in relaxation in both intact and denuded rings, with ouabain having little or no effect. Adenosine-induced relaxation appeared to involve the A2a receptor, but not the A2b subtype.ConclusionsIt was concluded that adenosine relaxation in NE-precontracted 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, SarcKATP and MitoKATP channels. The possible in vivo significance of the regulation of arterial compliance to left ventricular function coupling is discussed.

Effects and mechanisms of curcumin on the hemodynamic variablesof isolated perfused rat hearts.

There is no information on the dose-response relationship of curcumin on the hemodynamic variables of the heart at the organ level in isolated perfused rat hearts. We aimed to investigate the effects and mechanisms of curcumin on the hemodynamic variables of isolated perfused rat hearts. Rats were randomly divided into 9 groups. The isolated rat heart was retrogradely perfused with modified Krebs-Henseleit solution. After the stabilization period, each group was administered one of the following treatments for 25 min: saline, dimethyl sulfoxide, and curcumin (0.2 µM, 1 µM, and 5 µM); atropine (1 µM); atropine (1 µM) + curcumin (1 µM); L-NAME (100 µM); or L-NAME (100 µM) + curcumin (1 µM). Hemodynamic variables of the heart were measured. Curcumin at dose of 1 µM decreased the heart rate (from 271 ± 11.1 to 200.4 ± 14.3 beats/min, P = 0.011) but increased end-diastolic pressure (from 7.0 ± 0.4 to 54.6 ± 7.9 mmHg, P = 0.0008). A dose of 5 µM curcumin caused a decrease in the developed pressure (from 87.58 ± 9.0 to 65.40 ± 7.0 mmHg, P = 0.047) but an increase in the end-diastolic pressure (from 6.8 ± 0.6 to 48.9 ± 7.7 mmHg, P = 0.005). Atropine (1 µM) reversed the effects of curcumin on the heart. Our results suggest that curcumin produces dose-dependent negative chronotropic and inotropic effects in isolated perfused rat hearts.

Lipofundin® MCT/LCT 20% increase left ventricular systolic pressure in anex vivorat heart model via increase of intracellular calcium level

BackgroundLipid emulsions have been used to treat various drug toxicities and for total parenteral nutrition therapy. Their usefulness has also been confirmed in patients with local anesthetic-induced cardiac toxicity. The purpose of this study was to measure the hemodynamic and composition effects of lipid emulsions and to elucidate the mechanism associated with changes in intracellular calcium levels in myocardiocytes.MethodsWe measured hemodynamic effects using a digital analysis system after Intralipid® and Lipofundin® MCT/LCT were infused into hearts hanging in a Langendorff perfusion system. We measured the effects of the lipid emulsions on intracellular calcium levels in H9c2 cells by confocal microscopy.ResultsInfusion of Lipofundin® MCT/LCT 20% (1 ml/kg) resulted in a significant increase in left ventricular systolic pressure compared to that after infusing modified Krebs-Henseleit solution (1 ml/kg) (P = 0.003, 95% confidence interval [CI], 2.4–12.5). Lipofundin® MCT/LCT 20% had a more positive inotropic effect than that of Intralipid® 20% (P = 0.009, 95% CI, 1.4–11.6). Both lipid emulsion treatments increased intracellular calcium levels. Lipofundin® MCT/LCT (0.01%) increased intracellular calcium level more than that of 0.01% Intralipid® (P < 0.05, 95% CI, 0.0–1.9).ConclusionsThese two lipid emulsions had different inotropic effects depending on their triglyceride component. The inotropic effect of lipid emulsions could be related with intracellular calcium level.

Acidosis reduces the function and expression of α1D-adrenoceptor in superior mesenteric artery of Capra hircus.

Objective:The objective of this study was to characterize the α1-adrenoceptor (α1-AR) subtypes and evaluate the effect of acidosis on α1-AR function and expression in goat superior mesenteric artery (GSMA).Materials and Methods:GSMA rings were mounted in a thermostatically controlled (37.0°C ± 0.5°C) organ bath containing 20 ml of modified Krebs-Henseleit solution, maintained at pHo of 7.4, 6.8, 6.0, 5.5, 5.0, and 4.5. Noradrenaline (NA)- and phenylephrine (PE)-induced contractile response was elicited in the absence or presence of endothelium and prazosin at pHo of 7.4, 6.0, and 5.0. The responses were recorded isometrically by an automatic organ bath connected to PowerLab and analyzed using Labchart 7.1.3 software. Expression of α1D-AR was compared at physiological and acidic pHo using reverse transcription-polymerase chain reaction (RT-PCR).Results:NA- and PE-induced contractile responses were attenuated proportionately with a decrease in extracellular pH (pHo), i.e. 7.4 → 6.8 → 6.0 → 5.5 → 5.0 → 4.5. Endothelium denudation increased the contractile response at both normal and acidic pHo. Prazosin (1 nM, 10 nM, and 0.1 μM) inhibited the NA- and PE-induced contractile response at pHo 7.4 and the blocking effect of prazosin was potentiated at pHo of 6.0 and 5.0. RT-PCR analysis for α1D-AR in GSMA showed that the mRNA expression of α1D-AR was decreased under acidic pHo as compared to physiological pHo.Conclusion:(i) Adrenergic receptor mediates vasoconstriction in GSMA under normal physiological pHo, and α1D is the possible subtype involved in this event (ii) acidosis attenuates the vasocontractile response due to reduced function and expression of α1D-AR and also increased the release of endothelial-relaxing factors.

Abstract 245: Prevention of Glyceryl Trinitrate Tolerance by Flavonoids

Background and propose: The anti-ischemic effects of glyceryl trinitrate (GTN) are rapidly blunted after continuous application. This phenomenon of GTN tolerance may be the result of an increased oxidative stress and/or a reduced bioactivation of GTN by the enzyme aldehyde dehydrogenase (ALDH). The polyphenolic compounds flavonoids are reported to potentiate vascular relaxation and inhibit oxidation. Therefore, in the present study, we assessed the hypothesis that flavonoids can prevent GTN tolerance. Whether or not this effect of flavonoids is related to their anti-oxidative property or modulation of the activity of ALDH was also examined. Experimental approach: Isolated rat aortic rings were incubated with modified Krebs-Henseleit solution containing GTN (30 μM) in organ chamber at 37°C for 1 hour to induce tolerance. Flavonoids (10 μM of apigenin, luteolin, kaempferol, quercetin, genistin, genistein, narigenin, catechin or epicatechin) or the vehicle (1 mM dimethyl sulfoxide) were added to the aortic rings 30 minutes before GTN treatment. Those rings without incubation with GTN were served as the respective control groups. After removing the treatment solution, rings were contracted with phenylephrine (1 μM) followed by cumulative addition of GTN (0.1 nM to 30 μM). GTN-induced relaxation was calculated as change in isometric tension of aortic rings from the contraction induced by phenylephrine. Vascular ALDH activity was assayed by spectrophotometry as the rate of NAD+ consumption. Key results: GTN-induced relaxation in rat aortic rings was reduced by prior incubation with GTN; this reduction was prevented by apigenin (10 μM), which did not affect the relaxations in the group without prior treatment with GTN. Apigenin (10 μM) also increased the vascular ALDH activity which was reduced by prior GTN incubation. Conclusion: Apigenin appears to have the potential to prevent the development of GTN tolerance, likely by restoring the bioactivation of GTN by ALDH.

Evaluation of general toxicity, anti-oxidant activity and effects of ficus carica leaves extract on ischemia/reperfusion injuries in isolated heart of rat.

This study was aimed to evaluate general toxicity, anti-oxidant activity and effects of Ficus carica leaves extract on ischemia/reperfusion injuries. Antioxidant activity, total phenolic and flavonoid compounds of 70% methanolic extract of Ficus carica leaves were measured. The general toxicity test was carried out by brine shrimp lethality assay. Isolated hearts of male rats were mounted on a Langendorff apparatus and perfused with modified Krebs-Henseleit solution. In control group, the hearts were perfused with normal Krebs solution, however, treatment groups received enriched solution with the extract (0.04, 0.2 and 1 mg/ml) during stabilization and reperfusion (after 30 min global ischemia), respectively. Cardiac arrhythmias were analyzed and TTC method was used for infarct size determination. The extract displayed antioxidant activity in the DPPH assay (RC50=0.06666 mg/ml). Total phenolic content was 12.29 mg GAE/100 g dry sample and the amount of flavonoids was calculated 40.729 mg/g. LD50 value by brine shrimp test was 0.158 mg/ml. The extract decreased number of VEBs, incidence and duration of Rev VF with clear reduction in infarct size and infarct volume (P<0.001). Ficus carica decreased ischemia/reperfusion-induced injuries. These protections are probably due to antioxidant capacity and the existence of flavonoid and phenolic compounds in the extract.