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G A Gintant

Publications and source records attributed to G A Gintant.

At least 19 recordsLinked to original sources
PubMed2005

Pharmacological characterization of P2X7 receptors in rat peritoneal cells.

OBJECTIVE AND DESIGN: P2X(7) receptor activation by ATP results in the release of IL-1beta and IL-18. Prolonged stimulation can lead to pore formation and cell death. In this study we pharmacologically characterized P2X(7) receptors on rat peritoneal cells (RPC) and on 1321N1 cells transfected with rat P2X(7) receptor (1321rP2X(7)-11). MATERIALS AND METHODS: RPC were isolated from rats by lavage. P2X(7) agonist induced pore formation in RPC was measured by EtBr uptake. P2X(7)-stimulated pore formation and Ca(++) influx in 1321rP2X(7)-11 cells were measured by a fluorometric imaging plate reader. The effects of pyridoxal phosphate-6-azo phenyl -2'-4'-disulfonic acid (PPADS) on pore formation and Ca(++) influx were examined in both RPC and 1321rP2X(7)-11. P2X(7)-mediated IL-1beta release in RPC and the effect of PPADS were determined. RESULTS: RPC express functional P2X(7) receptors that were activated by ATP analogs with a rank order of potency of 2'- 3'-O-(4-Benzoylbenzoyl) adenosine 5'-triphosphate (BzATP) > ATP > alpha,beta-methylene ATP. Activation of P2X(7) receptors by BzATP was inhibited by PPADS. Similar results were also obtained in 1321rP2X(7)-11 cells. Activation of P2X(7) receptors on RPC resulted in IL-1 beta secretion, which was inhibited by PPADS. CONCLUSIONS: RPC express functional P2X(7) receptors that form pores and mediate the release of IL-1beta.

Adenosine Triphosphate↗
PubMed2001

The canine Purkinje fiber: an in vitro model system for acquired long QT syndrome and drug-induced arrhythmogenesis.

Torsade de pointes is a rare but potentially fatal ventricular arrhythmia associated with drug-induced delayed repolarization and prolongation of the QT interval. To determine if the arrhythmogenic potential of noncardiac drugs can be assessed in vitro, we evaluated the effects of 12 drugs on the action potential duration (APD) of cardiac Purkinje fibers and compared results with clinical observations. APD changes in canine and porcine fibers were evaluated under physiologic conditions (37 degrees C, [K+]0 = 4 mM) using standard microelectrode techniques. Six of seven drugs associated with QT prolongation or torsade de pointes in man (cisapride, erythromycin, grepafloxacin, moxifloxacin, sertindole, and sotalol) affected concentration-dependent prolongation of the APD in canine fibers during slow stimulation (2-s basic cycle length), attaining greater than 15% prolongation at high concentrations (> or = 10-fold clinically encountered plasma levels). Each of five drugs not linked clinically to QT prolongation and torsade de pointes (azithromycin, enalaprilat, fluoxetine, indomethacin, and pinacidil) failed to attain 15% prolongation, with fluoxetine, indomethacin, and pinacidil abbreviating the APD. Drugs eliciting the greatest prolongation also demonstrated prominent reverse rate-dependent effects. The antihistamine terfenadine (linked to dose-dependent QT prolongation and torsade de pointes clinically) only minimally prolonged the APD in canine and porcine fibers (and exerted no effect on midmyocardial fibers from left ventricular free wall) at supratherapeutic concentrations. On the basis of concentration-dependent APD prolongation and reverse rate-dependent effects, this Purkinje fiber model detects six of seven drugs linked clinically to acquired long QT syndrome and torsade de pointes, and clears each of five drugs not associated with repolarization abnormalities (overall 92% accuracy), validating the utility of this Purkinje fiber model in the preclinical evaluation of QT prolongation and proarrhythmic risk by noncardiac drugs.

Action Potentials↗
PubMed2000

Characterization and functional consequences of delayed rectifier current transient in ventricular repolarization.

Although inactivation of the rapidly activating delayed rectifier current (I(Kr)) limits outward current on depolarization, the role of I(Kr) (and recovery from inactivation) during repolarization is uncertain. To characterize I(Kr) during ventricular repolarization (and compare with the inward rectifier current, I(K1)), voltage-clamp waveforms simulating the action potential were applied to canine ventricular, atrial, and Purkinje myocytes. In ventricular myocytes, I(Kr) was minimal at plateau potentials but transiently increased during repolarizing ramps. The I(Kr) transient was unaffected by repolarization rate and maximal after 150-ms depolarizations (+25 mV). Action potential clamps revealed the I(Kr) transient terminating the plateau. Although peak I(Kr) transient density was relatively uniform among myocytes, potentials characterizing the peak transients were widely dispersed. In contrast, peak inward rectifier current (I(K1)) density during repolarization was dispersed, whereas potentials characterizing I(K1) defined a narrower (more negative) voltage range. In summary, rapidly activating I(Kr) provides a delayed voltage-dependent (and functionally time-independent) outward transient during ventricular repolarization, consistent with rapid recovery from inactivation. The heterogeneous voltage dependence of I(Kr) provides a novel means for modulating the contribution of this current during repolarization.

Action Potentials↗
PubMed1998

Azimilide causes reverse rate-dependent block while reducing both components of delayed-rectifier current in canine ventricular myocytes.

Most class III antiarrhythmic drugs reduce the rapidly activating component of delayed-rectifier current (IKr) without affecting the slowly activating component (IKs). Recently the novel antiarrhythmic agent azimilide (NE-10064) was reported to enhance IKs at low (nanomolar) concentrations and to block both IKr and IKs at higher (micromolar) concentrations. Further to understand the electrophysiologic effects of azimilide, we compared its effects on IKr and IKs (by using whole cell clamp techniques) and action potentials (microelectrode and perforated-patch techniques) on canine ventricular myocytes. A lower azimilide concentration (50 nM) did not enhance IKs. In contrast, a therapeutic azimilide concentration (2 microM) was equieffective in reducing IKr (300-ms isochrones) and IKs (3-s isochrones) by approximately 40% during depolarizing test pulses, as well as reducing IKr (38% decrease) and IKs (33% decrease) tail currents on repolarization. Block of IKs was independent of voltage at positive test potentials. In action-potential studies, 50 nM azimilide had no effect on the action-potential duration (APD), whereas 2 microM azimilide delayed repolarization and caused reverse rate-dependent effects on the APD. Whereas the extent of APD prolongation by azimilide was not correlated with the drug-free APD, azimilide preferentially exaggerated the APD-rate relationship of myocytes displaying the steepest APD-rate relationship under drug-free conditions. In conclusion, therapeutic concentrations of azimilide that cause comparable reduction of canine ventricular IKr and IKs exert reverse rate-dependent effects, which are dependent on the steepness of the APD-rate relationship.

Action Potentials↗
PubMed1997

High extracellular glucose impairs cardiac E-C coupling in a glycosylation-dependent manner.

Hyperglycemia is a major manifestation of all forms of diabetes mellitus and is associated with increased risk of cardiovascular disease. It is well established that cardiac excitation-contraction (E-C) coupling is adversely affected in diabetic animals such that ventricular myocyte action potential duration is prolonged and intracellular Ca2+ clearing and mechanical relaxation are slowed. We now report that ventricular myocytes incubated in a culture medium containing high extracellular glucose (25.5 mM) also exhibit these same changes in E-C coupling. These effects are not manifested for approximately 24 h after exposure. Furthermore, in the presence of normal glucose (5.5 mM), relaxation is also prolonged by fructose (20 mM), yet is unaffected by equimolar concentrations of nonmetabolizable sugars such as L-glucose and mannitol, implying that the high glucose effects require glucose entry into the cell and metabolic processing. The prolonged relaxation can also be produced by 5 mM glucosamine (an intermediate of glycosylation) and is blocked by 0.5 microgram/ml tunicamycin (an inhibitor of N-linked glycoprotein synthesis). Culturing myocytes with an inhibitor of glycation (10 mM aminoguanidine) does not prevent the high extracellular glucose concentration effects. Thus our data indicate that high extracellular glucose impairs cellular mechanisms contributing to myocardial relaxation and that this impairment may involve glycosylation of nascent proteins.

Action Potentials↗
PubMed1997

Tissue and species distribution of mRNA for the IKr-like K+ channel, erg.

The human K+ channel gene, HERG, has been linked to the type 2 form of the autosomal dominant long-QT syndrome and has been suggested to encode the fast component of the delayed rectifier K+ current (IKr) found in heart. To date, the published electrophysiological and pharmacological data on the Xenopus-expressed HERG are very similar but are not identical to those of the endogenous IKr. In an effort to provide a different type of correlative data on the relationship between erg and IKr. cDNA fragments of erg homologues from guinea pig, rabbit, human, dog, and rat were cloned and used to test for the presence of erg mRNA in cardiac tissue. RNase protection assays reveal that erg message is found in the hearts of all five species and that it is expressed uniformly throughout the heart. The erg transcript is expressed at relatively high levels, being approximately 50% more abundant than the most prevalent Kv-class K+ channel transcript in canine ventricle (Kv4.3) erg transcripts were found to have a wide tissue distribution in rat and are abundant in the brain, retina, thymus, and adrenal gland and are also found in skeletal muscle, lung, and cornea. Since there were no published reports of an IKr-like current in the rat heart, electrophysiological studies were performed to test whether the significant level of erg message in rat heart was correlated with the presence of an IKr-like current in rat. In isolated rat ventricular myocytes, an E-4031-sensitive current was observed, which is consistent with the presence of IKr. These results strengthen the link between erg and the native IKr in heart and suggest that erg may play an important role in other noncardiac tissues.

Amino Acid Sequence↗
PubMed1996

Two components of delayed rectifier current in canine atrium and ventricle. Does IKs play a role in the reverse rate dependence of class III agents?

Because the number and characteristics of delayed rectifier K+ current (IK) components vary between species, the role of each component in the action potential and modulation by class III agents is uncertain. To address these issues, IK was assessed in adult isolated canine ventricular and and atrial myocytes by using whole-cell and perforated-patch techniques. IK components were characterized by using two complementary approaches: a kinetic approach (based on biexponential fits to deactivating tail currents) and a pharmacological approach approach (using the methanesulfonanilide compound E-4031). In ventricular myocytes, two exponential tail current components were distinguished; these components differed in the voltage and time dependence of activation and the effect of lower (K+). Both kinetic components contributed equally to peak tail current amplitude (measured at -35 mV) after a single 300-ms pulse to 5 mV, simulating an action potential. By use of E-4031, rapidly and slowly activating components described kinetically were identified. The activation kinetics and rectification properties of canine IKr and IKs are qualitatively similar to those described previously for guinea pigs. In contrast, canine IKr and IKs deactivation kinetics differed markedly from those found in guinea pigs, with canine IKr deactivating slowly (time constant tau, 2 to 3 s near -35 mV) and IKs deactivating rapidly (tau, 150 ms near -35 mV and decreasing to 30 ms near -85 mV). E-4031 elicited reverse rate-dependent effects (greater drug-induced prolongation of the action potential at slower stimulation rates); this effect is inconsistent with the hypothesis attributing reverse rate dependence to incomplete IKs deactivation during rapid stimulation (due to rapid deactivation of canine IKs). Two IK components with characteristics comparable to those found in ventricular myocytes were also observed in atrial myocytes. In conclusion, (1) IKr- and IKs-like components of IK are present in canine atrial and ventricular myocytes, with deactivation kinetics strikingly different from those found in guinea pigs, and (2) the rapid deactivation kinetics of canine IKs do not support its role in reverse rate dependence with class III agents in this species.

Action Potentials↗
PubMed1995

Regional differences in IK density in canine left ventricle: role of IK,s in electrical heterogeneity.

Delayed rectifier K+ current (IK) was studied in isolated myocytes from canine left ventricular epicardium and midmyocardium using whole cell patch-clamp techniques. IK density during activation was greater in epicardial vs. midmyocardial myocytes [1.06 +/- 0.09 vs. 0.66 +/- 0.09 pA/pF (SE); P < 0.01] measured during 3-s depolarizing pulses to +25 mV. IK density was greater in epicardial myocytes at all times examined (range 150 ms to 3 s, +25 mV) and on termination of 3-s test pulses (potentials +5 to +65 mV). Greater IK density could not be explained by differences in activation kinetics or voltage dependence of activation. Two components of IK (IK,r and IK,s) have been described in guinea pig myocytes (Sanguinetti, M. C., and N. Jurkiewicz, J. Gen. Physiol. 96: 192-214, 1990). To assess whether differences in IK density could be ascribed to IK,r or IK,s, tail currents were fit to the sum of two decaying exponentials, with each component analogous to IK,s and IK,r based on sensitivity to E-4031 and rectification properties. Greater tail current density in epicardial myocytes was due to greater IK,s with no discernible difference in IK,r. These results suggest that regional differences in IK density in left ventricular epicardium are due to a larger IK,s component, which contributes to ventricular electrical heterogeneity and may reflect the differential expression of the IsK channel.

Animals↗
PubMed1993

Ionic bases for electrophysiological distinctions among epicardial, midmyocardial, and endocardial myocytes from the free wall of the canine left ventricle.

Recent studies from our laboratory involving syncytial preparations have delineated electrophysiological distinctions between epicardium, endocardium, and a unique population of cells in the deep subepicardial to midmyocardial layers (M region) of the canine ventricle. In the present study, we used standard microelectrode, single microelectrode switch voltage-clamp, and whole-cell patch-clamp techniques to examine transmembrane action potentials, steady-state current-voltage relations, and the 4-aminopyridine-sensitive transient outward current (Ito1) in myocytes enzymatically dissociated from discrete layers of the free wall of the canine left ventricle. Action potential characteristics of myocytes isolated from the epicardium, M region, and endocardium were very similar to those previously observed in syncytial preparations isolated from the respective regions of the ventricular wall. A prominent spike and dome was apparent in myocytes from epicardium and the M region but not in myocytes from endocardium. Action potential duration-rate relations were considerably more pronounced in cells isolated from the M region. Current-voltage relations recorded from cells of epicardial, M region, and endocardial origin all displayed an N-shaped configuration with a prominent negative slope-conductance region. The magnitude of the inward rectifier K+ current (IK1) was 392 +/- 86, 289 +/- 65, and 348 +/- 115 pA in epicardial, M region, and endocardial myocytes, respectively, when defined as steady-state current blocked by 10 mM Cs+. Similar levels were obtained when IK1 was defined as the steady-state difference current measured in the presence (6 mM) and absence of extracellular K+. Ito1 was significantly greater in epicardial and M region myocytes than in endocardial myocytes. At a test potential of +70 mV (holding potential, -80 mV), Ito1 amplitude was 4,203 +/- 2,370, 3,638 +/- 1,135, and 714 +/- 286 pA in epicardial, M region, and endocardial cells, respectively. No significant differences were observed in the voltage dependence of inactivation of Ito1 in the three cell types. The time course of reactivation of Ito1 was slower in cells from the M region compared with either epicardial or endocardial cells. Our data suggest that prominent heterogeneity exists in the electrophysiology of cells spanning the canine ventricular wall and that differences in the intensity of the transient outward current contribute importantly, but not exclusively, to this heterogeneity. These findings should advance our understanding of basic heart function and the ionic bases for the electrocardiographic J wave, T wave, U wave, and long QT intervals as well as improve our understanding of some of the complex factors contributing to the development of cardiac arrhythmias.

Action Potentials↗
PubMed1992

Beta-adrenergic modulation of fast inward sodium current in canine myocardium. Syncytial preparations versus isolated myocytes.

Reports have suggested that the fast inward sodium current (INa) in cardiac tissues may be modulated by beta-adrenergic stimulation and that such modulation may affect conduction in the setting of myocardial ischemia and infarction. However, many of these studies have used dissociated myocytes or broken cell preparations, whose responses need not necessarily reflect those of syncytial preparations. To investigate further the possibility that beta-adrenergic stimulation of INa may differ in various preparations, we compared the effects of the beta-agonist isoproterenol (ISO) on syncytial canine Purkinje fibers and ventricular muscle preparations, as well as isolated ventricular myocytes. Alterations of the maximum rate of rise of the action potential upstroke (Vmax) were used as an index of changes of INa. ISO (1 microM) had no effect on Vmax of upstrokes of normally polarized (fast responses) or partially depolarized (elevated [K+]o, depressed fast responses) syncytial ventricular muscle preparations or Purkinje fibers. In contrast, lower concentrations of ISO (0.5-1.0 microM) modulated Vmax of isolated ventricular myocytes, depending on the technique used to monitor transmembrane potential. When 2.7 M KCl-filled microelectrodes were used, ISO reduced Vmax of partially depolarized myocytes without affecting Vmax of normally polarized myocytes. However, when myocytes were dialyzed using patch pipettes, ISO reduced Vmax of partially depolarized myocytes and increased Vmax of normally polarized myocytes, effecting a hyperpolarized shift of the normalized inactivation curve relating Vmax to resting membrane potential. The different beta-adrenergic responses of syncytial preparations and nondialyzed and dialyzed myocytes suggest that differences in the ionic or metabolic condition of the preparations likely alter cAMP-dependent responses and channel phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Action Potentials↗
PubMed1987

Properties of an electrogenic sodium-potassium pump in isolated canine Purkinje myocytes.

1. Purkinje myocytes were isolated from canine Purkinje strands by collagenase exposure and gentle trituration. The myocytes were studied by a switched single-micro-electrode voltage-clamp technique at 37 degrees C in Tyrode solution containing 8 mM-K+ and 2 mM-Ca2+. 2. The dose-response relation for the cardiotonic steroid dihydroouabain (DHO) was obtained by measuring the change in membrane current caused by application of concentrations of 1-100 microM. The KD obtained in fourteen experiments was 3.7 +/- 1.1 microM (mean +/- S.E. of mean). 3. We employed 100 microM-DHO (a concentration more than 25-fold greater than the KD) to estimate the resting pump current (Ip) in the isolated myocytes. A value of 0.27 +/- 0.02 microA microF-1 (mean +/- S.E. of mean, n = 32) was obtained. 4. Myocytes were also exposed to K+-free solution for a period of 200 s. On return to K+-containing Tyrode solution there was a slowly decaying outward current. The time constant of decay of this pump current transient was 87 +/- 8 s (mean +/- S.E. of mean, n = 8). The integral beneath this transient was used to obtain a second estimate of the resting pump current. In four preparations where exposures in DHO and in K+-free solutions were employed the ratio Ip, DHO/Ip, K-free was 1.76 +/- 0.15 (mean +/- S.E. of mean). 5. From the magnitude of resting pump current, in the presence of total pump blockade the Na+ activity should rise at a rate of 1.3 mM min-1. 6. Reducing [K+]o from 8 to 1 mM reduced Ip by more than 40% initially. Ip then slowly increased over the next 30 min. These results suggest that the steady-state inward background current is not greatly altered by changes in [K+]o, and that [Na+]i rises to a new level. The changes in Ip obtained at early times following reduction of [K+]o to 1 or 0.5 mM (t less than 1.75 min) were used to estimate the Km for external K+; a value of 0.8 mM was obtained. 7. The results suggest that the properties of the Na+-K+ pump in isolated canine Purkinje myocytes are similar to those in canine Purkinje strands. This argues against major distortions of measured pump properties in the canine Purkinje strand and for the physiological state of the Na+-K+ pump in the isolated Purkinje myocyte.

Animals↗
PubMed1985

Microprocessor controlled trituration device for the dissociation of cardiac and other tissues.

The construction of an inexpensive microprocessor controlled piston/cylinder used in the dissociation of canine cardiac Purkinje fibres is described. The microprocessor, interfaced to a linear actuator, controls the motion of a piston using the higher level language "Tiny BASIC" and two machine language routines. Pressure changes generated by the piston are transferred to the lumen of a pipette. Movement of enzymatically-treated tissues through the pipette orifice facilitates dissociation into single cells. With this device, specific agitation protocols for different quantities and types of tissues are possible.

Animals↗
PubMed1985

Versatile temperature controlled tissue bath for studies of isolated cells using an inverted microscope.

The design of a small, inexpensive temperature controlled bath (0.25 ml volume) for electrophysiological studies of isolated cells is described. The design creates a uniform temperature (+/- 1 degree C) from ambient temperature to approximately 37 degrees C over a bath chamber area of 0.6 X 1 cm for flow rates from zero to 2 ml/min. Access for microelectrodes and unobstructed preparation viewing with an inverted microscope is afforded by a finely stranded heater sandwiched between two glass cover slips and located beneath the bath chamber. Battery operation of the feedback temperature controller and solution pre-heater minimizes electrical interference.

Cytological Techniques↗
PubMed1985

Gating of delayed rectification in acutely isolated canine cardiac Purkinje myocytes. Evidence for a single voltage-gated conductance.

Studies of time-dependent, plateau outward current (delayed rectification) in the heart are complicated by the accumulation and depletion of K+ ions in intercellular clefts. To minimize this problem, we studied delayed rectification in acutely isolated (enzymic solution, gentle agitation) canine cardiac Purkinje myocytes using the single microelectrode voltage-clamp technique. We found a sigmoidal voltage-dependence for activation of outward plateau current, with maximal activation occurring at potentials near -10 mV. The activation and deactivation of plateau outward current was adequately described as the sum of a fast and slow exponential component. A comparison of the time course of activation of plateau outward current and the "envelope" of tail currents suggests that a single voltage-gated conductance with one open and two closed states can account for delayed rectification in Purkinje myocytes. These results differ from those previously obtained with intact sheep Purkinje fibers in which two time-dependent conductances were postulated to account for delayed rectification (Noble, D., and R. W. Tsien, 1969, J. Physiol. (Lond.), 200:205-231).

Action Potentials↗
PubMed1984

Use-dependent block of cardiac sodium channels by quaternary derivatives of lidocaine.

Modulation of the reduction of fast inward sodium current by local anesthetics due to changes in electrical activity has been termed use-dependent block ( Courtney 1975). To determine the mechanisms responsible for use-dependent block of cardiac sodium channels and to compare use-dependent block in cardiac and nerve preparations, we investigated use-dependent block of cardiac sodium channels by the quaternary lidocaine analogues QX -314 and QX -222 (two agents previously studied in nerve). We used canine cardiac Purkinje fibers, and assessed changes in the fast inward sodium current using changes in the maximum rate of rise of the action potential upstroke (Vmax). Two microelectrode voltage clamp and current clamp techniques were used to control membrane potential prior to stimulated upstrokes . Use-dependent block was not affected by shortening the action potential duration during rapid stimulation. Partial recovery from use-dependent block was observed during rapid stimulation with brief depolarizing prepulses terminating immediately prior to the upstroke. Similar prepulses also prevented the development of use-dependent block following an abrupt increase in the stimulation rate. Hyperpolarizing prepulses during rapid stimulation caused recovery from use-dependent block; recovery was greater and more rapid with increasingly negative prepulses . Hyperpolarization during periods of electrical quiescence also caused greater recovery. These results, interpreted using the modulated receptor hypothesis ( Hille 1977; Hondeghem and Katzung 1977), suggest that use-dependent block of cardiac sodium channels by quaternary local anesthetics is due to drug association with the inactivated sodium channel receptor which occurs only after these drugs gain access to the receptor site through open sodium channels.

Action Potentials↗