A method for making rapid changes of superfusate whilst maintaining temperature at 37°C

Levi, A J; Hancox, JC; Howarth, FC; Croker, J; Vinnicombe, J

doi:10.1007/s004240050217

Cited by 72 publications

(55 citation statements)

References 14 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…At 10, 40, 55, 70, and 80 min during the experiment, stimulation was briefly interrupted, and 10 mM caffeine was applied to cells for 1 s at 37°C with a rapid solution changer. Changes in solution with this device were triggered by the voltage-clamp protocol and were complete within 300 ms (27). ]i were recorded at 5-min intervals throughout the experiment except for the first 5 min of reperfusion, when recordings were made every minute.…”

Section: Methodsmentioning

confidence: 99%

Changes in excitation-contraction coupling in an isolated ventricular myocyte model of cardiac stunning

Louch

Ferrier

Howlett

2002

American Journal of Physiology-Heart and Circulatory Physiology

View full text Add to dashboard Cite

To investigate cardiac stunning, we recorded intracellular [Ca2+], contractions, and electrical activity in isolated guinea pig ventricular myocytes exposed to simulated ischemia and reperfusion. After equilibration, ischemia was simulated by exposing myocytes to hypoxia, acidosis, hyperkalemia, hypercapnia, lactate accumulation, and substrate deprivation for 30 min at 37°C. Reperfusion was simulated by exposure to Tyrode solution. Field-stimulated myocytes exhibited stunning upon reperfusion. By 10 min of reperfusion, contraction amplitude decreased to 43.0 ± 5.5% of preischemic values ( n = 15, P < 0.05), although action potential configuration and sarcoplasmic reticulum Ca2+stores, assessed with caffeine, were normal. Diastolic [Ca2+] and Ca2+ transients (fura 2) were also normal in stunned myocytes. In voltage-clamped cells, peak L-type Ca2+ current was reduced to 47.4 ± 4.5% of preischemic values at 10 min of reperfusion ( n= 21, P < 0.05). Contractions elicited by Ca2+-induced Ca2+ release and the voltage-sensitive release mechanism were both depressed in reperfusion. Our observations suggest that stunning is associated with reduced L-type Ca2+ current but that alterations in Ca2+ homeostasis and release are not directly responsible for stunning.

show abstract

Section: Methodsmentioning

confidence: 99%

Changes in excitation-contraction coupling in an isolated ventricular myocyte model of cardiac stunning

Louch

Ferrier

Howlett

2002

American Journal of Physiology-Heart and Circulatory Physiology

View full text Add to dashboard Cite

show abstract

“…• C and loaded with fura-2, stimulation was paused for a period of 5 s. Caffeine (20 mM) was then applied for 10 s using a solutionswitching device customized for rapid solution exchange (Levi et al 1996). Electrical stimulation was then resumed, was assessed by measuring the area under the curve of the caffeine-evoked Ca 2+ transient.…”

Section: Measurement Of Sarcoplasmic Reticulum Ca 2+ Transportmentioning

confidence: 99%

Structural lesions and changing pattern of expression of genes encoding cardiac muscle proteins are associated with ventricular myocyte dysfunction in type 2 diabetic Goto-Kakizaki rats fed a high-fat diet

Howarth¹,

Qureshi²,

Sobhy³

et al. 2011

Experimental Physiology

View full text Add to dashboard Cite

Given the clinical prevalence of type 2 diabetes and obesity and their association with high mortality linked to cardiovascular disease, the aim of the study was to investigate the effects of feeding type 2 diabetic Goto-Kakizaki (GK) rats either high-or low-fat diets on cardiomyocyte structure and function. The GK rats were fed either a high-fat diet (HFD) or a low-fat diet (LFD) from the age of 2 months for a period of 7 months. The GK-HFD rats gained more weight, ate less food and drank less water compared with GK-LFD rats. At 7 months, non-fasting blood glucose was higher in GK-LFD (334 ± 35 mg dl −1 ) compared with GK-HFD rats (235 ± 26 mg dl −1 ). Feeding GK rats with a HFD had no significant effect on glucose clearance following a glucose challenge. Time-to-peak (t peak ) shortening was reduced in myocytes from GK-HFD (131.8 ± 2.1 ms) compared with GK-LFD rats (144.5 ± 3.0 ms), and time-to-half (t 1 /2 ) relaxation of shortening was also reduced in myocytes from GK-HFD (71.7 ± 6.9 ms) compared with GK-LFD rats (86.1 ± 3.6 ms). The HFD had no significant effect on the amplitude of shortening. The HFD had no significant effect on t peak , t 1 /2 decay, amplitude of the Ca 2+ transient, myofilament sensitivity to Ca 2+ , sarcoplasmic reticulum Ca 2+ content, fractional release of Ca 2+ and the rate of Ca 2+ uptake. Structurally, ventricular myocytes from GK-HFD rats showed extensive mitochondrial lesions, including swelling, loss of cristae, and loss of inner and outer membranes, resulting in gross vacuolarization and deformation of ventricular mitochondria with a subsequent reduction in mitochondrial density. Expression of genes encoding various L-type Ca 2+ channel proteins (Cacnb2) and cardiac muscle proteins (Myl2 and Atp2a1) were downregulated in GK-HFD compared with GK-LFD rats. Structural lesions and changed expression of genes encoding various cardiac muscle proteins might partly underlie the altered time course of myocyte shortening and relaxation in myocytes from GK-HFD compared with GK-LFD rats. A variety of diastolic and systolic dysfunctions have been widely reported in type 2 diabetic patients, and the severity of abnormalities depends on the patients' age and the duration of diabetes. Haemodynamic abnormalities include reduced left ventricular ejection fraction, impaired myocardial velocity at early diastole, abnormal relaxation during the early filling phase, prolonged isovolumetric relaxation, lower peak systolic and early diastolic velocity, impaired diastolic relaxation and filling and reduced peak filling rate (von Bibra et al. 2005;Dounis et al. 2006;Markuszewski et al. 2006;Sharman et al. 2007). The GotoKakizaki (GK) rat is a genetic animal model of type 2 diabetes, which was created by selective breeding of an outbred colony of Wistar rats with selection for high

show abstract

“…A constant flow of superfusate was applied with a 'rapid solution switcher' (adapted for oocytes from Levi et al, 1996). Initial application of propafenone always began with exposure to the drug for 2 min while maintaining the holding potential of the oocytes at Ϫ90 mV.…”

Section: Measurements Of Heterologous Herg Currents Isolation Ofmentioning

confidence: 99%

The Low-Potency, Voltage-Dependent HERG Blocker Propafenone—Molecular Determinants and Drug Trapping

Witchel

Dempsey²,

Sessions³

et al. 2004

Molecular Pharmacology

Self Cite

103

104

View full text Add to dashboard Cite

The molecular determinants of high-affinity human ether-a-gogo-related gene (HERG) potassium channel blockade by methanesulfonanilides include two aromatic residues (Phe656 and Tyr652) on the inner helices (S6) and residues on the pore helices that face into the inner cavity, but determinants for lower-affinity HERG blockers may be different. In this study, alanine-substituted HERG channel mutants of inner cavity residues were expressed in Xenopus laevis oocytes and were used to characterize the HERG channel binding site of the antiarrhythmic propafenone. Propafenone's blockade of HERG was strongly dependent on residue Phe656 but was insensitive or weakly sensitive to mutation of Tyr652, Thr623, Ser624, Val625, Gly648, or Val659 and did not require functional inactivation. Homology models of HERG based on KcsA and MthK crystal structures, representing the closed and open forms of the channel, respectively, suggest propafenone is trapped in the inner cavity and is unable to interact exclusively with Phe656 in the closed state (whereas exclusive interactions between propafenone and Phe656 are found in the open-channel model). These findings are supported by very slow recovery of wild-type HERG channels from block at Ϫ120 mV, but extremely rapid recovery of D540K channels that reopen at this potential. The experiments and modeling suggest that the open-state propafenone binding-site may be formed by the Phe656 residues alone. The binding site for propafenone (which may involve -stacking interactions with two or more Phe656 side-chains) is either perturbed or becomes less accessible because of closed-channel gating. This provides further evidence for the existence of gating-induced changes in the spatial location of Phe656 side chains.Pharmacological blockade of the cardiac 'rapid' delayed rectifier potassium (K ϩ ) current (I Kr ) is commonly associated with a drug's propensity to cause acquired long QT syndrome (Roden et al., 1996). The ␣-subunit of the I Kr channel is encoded by HERG (Sanguinetti et al., 1995). Molecular determinants of this channel's blockade by the archetypal highpotency HERG blockers, the methanesulfonanilides, reside in the inner cavity of the channel pore and are composed of amino acid residues in the H5 loop (adjacent to the selectivity filter) and the S6 transmembrane domain (Lees-Miller et al., 2000;Mitcheson et al., 2000a). For all drugs tested, the molecular determinants include the amino acid residues Phe656 and Tyr652 [except for vesnarinone, which does not require Tyr652 (Kamiya et al., 2001), and fluvoxamine, which can block HERG when Phe656 or Tyr652 are mutated ], and in many cases the molecular determinants additionally include a combination of Thr623, Ser624, Val625, Gly648, and Val659. Most drugs with a high-affinity for HERG have been shown to have an open-state-dependent blockade mechanism (Spector et al., 1996). Open-state blockade of HERG may involve drug trapping [e.g., MK-499 (Mitcheson et al., 2000b)], a 'foot in the door' type mechanism [e.g., chloroquine (Sanch...

show abstract

A method for making rapid changes of superfusate whilst maintaining temperature at 37°C

Cited by 72 publications

References 14 publications

Changes in excitation-contraction coupling in an isolated ventricular myocyte model of cardiac stunning

Changes in excitation-contraction coupling in an isolated ventricular myocyte model of cardiac stunning

Structural lesions and changing pattern of expression of genes encoding cardiac muscle proteins are associated with ventricular myocyte dysfunction in type 2 diabetic Goto-Kakizaki rats fed a high-fat diet

The Low-Potency, Voltage-Dependent HERG Blocker Propafenone—Molecular Determinants and Drug Trapping

Contact Info

Product

Resources

About