Atsuko Yatani

Dual effects of dihydropyridines on whole cell and unitary calcium currents in single ventricular cells of guinea‐pig.

We studied the effects of dihydropyridine Ca channel ligands (DHPs), mainly nitrendipine and Bay K8644, on whole cell and single channel Ca currents on single myocytes isolated from the adult guinea‐pig ventricle. Nitrendipine had dual effects, stimulatory or inhibitory, depending upon the membrane potential. At low frequencies (less than 0.03 Hz) and negative holding potentials (‐90 mV or more), nitrendipine increased the Ca currents in a dose‐dependent manner. The dose‐response curve was best fitted by a Langmuir adsorption isotherm model which was the sum of two independent one‐to‐one drug‐receptor sites with median effective doses (ED50S) of 1.0 X 10(‐9) M and 1.4 X 10(‐6) M respectively. When the membrane potential was held at ‐30 mV or less, nitrendipine inhibited the Ca currents, also in a dose‐dependent manner. The dose‐response curve was fitted by a single binding site model having a median inhibitor concentration (IC50) of 1.5 X 10(‐9) M. At holding potentials between ‐70 and ‐40 mV, nitrendipine produced mixed effects on Ca currents; an increase occurred initially and this was followed by a decrease. When rundown was excluded, Bay K8644 showed only stimulatory effects on the Ca currents between holding potentials of ‐120 and ‐30 mV. When the test potential was zero or +10 mV the Ca currents reached peak values and the dose‐response curve was best fitted by a single binding site model having an ED50 of 3 X 10(‐8) M. When the effects were measured at negative test potentials of ‐30 to ‐10 mV, the curve was best fitted by a two‐site model with ED50S of 3 X 10(‐9) and 9 X 10(‐7) M. At the single Ca channel level the stimulatory effect of nitrendipine was due to an increased probability that a Ca channel which had opened once would reopen, a reduction in records without activity and an increase in the mean open time. There were no changes in unit conductance. Inhibitory effects were due to a large increase in nulls. At lower concentrations the main effect of Bay K8644 was an increase in the probability of opening. At doses above 10(‐6) M, a pronounced increase in the open time was observed. The effects we observed are attributed to at least two sites for DHP related to Ca channels; one with high affinity and one with a lower affinity. The low affinity site mediates a stimulatory effect due to greatly prolonged openings.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibition of Glycogen Synthase Kinase 3β During Heart Failure Is Protective

Glycogen synthase kinase (GSK)-3, a negative regulator of cardiac hypertrophy, is inactivated in failing hearts. To examine the histopathological and functional consequence of the persistent inhibition of GSK-3&bgr; in the heart in vivo, we generated transgenic mice with cardiac-specific overexpression of dominant negative GSK-3&bgr; (Tg-GSK-3&bgr;-DN) and tetracycline-regulatable wild-type GSK-3&bgr;. GSK-3&bgr;-DN significantly reduced the kinase activity of endogenous GSK-3&bgr;, inhibited phosphorylation of eukaryotic translation initiation factor 2Bϵ, and induced accumulation of &bgr;-catenin and myeloid cell leukemia-1, confirming that GSK-3&bgr;-DN acts as a dominant negative in vivo. Tg-GSK-3&bgr;-DN exhibited concentric hypertrophy at baseline, accompanied by upregulation of the &agr;-myosin heavy chain gene and increases in cardiac function, as evidenced by a significantly greater Emax after dobutamine infusion and percentage of contraction in isolated cardiac myocytes, indicating that inhibition of GSK-3&bgr; induces well-compensated hypertrophy. Although transverse aortic constriction induced a similar increase in hypertrophy in both Tg-GSK-3&bgr;-DN and nontransgenic mice, Tg-GSK-3&bgr;-DN exhibited better left ventricular function and less fibrosis and apoptosis than nontransgenic mice. Induction of the GSK-3&bgr; transgene in tetracycline-regulatable wild-type GSK-3&bgr; mice induced left ventricular dysfunction and premature death, accompanied by increases in apoptosis and fibrosis. Overexpression of GSK-3&bgr;-DN in cardiac myocytes inhibited tumor necrosis factor-&agr;–induced apoptosis, and the antiapoptotic effect of GSK-3&bgr;-DN was abrogated in the absence of myeloid cell leukemia-1. These results suggest that persistent inhibition of GSK-3&bgr; induces compensatory hypertrophy, inhibits apoptosis and fibrosis, and increases cardiac contractility and that the antiapoptotic effect of GSK-3&bgr; inhibition is mediated by myeloid cell leukemia-1. Thus, downregulation of GSK-3&bgr; during heart failure could be compensatory.

Profile of the oppositely acting enantiomers of the dihydropyridine 202-791 in cardiac preparations: receptor binding, electrophysiological, and pharmacological studies.

Receptor binding, electrophysiological, and inotropic effects of the pure dihydropyridine enantiomers (+)S202-791 and (-)R202-791 were studied in cardiac preparations. The KI for (+)S202-791 binding correlated with the ED50s for an increase in contractile force and an increase in calcium current, the latter effect occurring at depolarized as well as resting holding potentials. The KI for (-)R202-791 binding was much lower than the IC50s for inhibition of calcium current measured at holding potentials of -80 or -90 mV and a negative inotropic effect, but correlated closely with the IC50 for inhibition of calcium current measured at -30 mV. Thus, (+)S202-791, is a voltage independent calcium channel activator and (-)R202-791 is a voltage dependent calcium channel inhibitor.

Glycogen Synthase Kinase-3α Reduces Cardiac Growth and Pressure Overload-induced Cardiac Hypertrophy by Inhibition of Extracellular Signal-regulated Kinases

Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase having multiple functions and consisting of two isoforms, GSK-3α and GSK-3β. Pressure overload increases expression of GSK-3α but not GSK-3β. Despite our wealth of knowledge about GSK-3β, the function of GSK-3α in the heart is not well understood. To address this issue, we made cardiac-specific GSK-3α transgenic mice (Tg). Left ventricular weight and cardiac myocyte size were significantly smaller in Tg than in non-Tg (NTg) mice, indicating that GSK-3α inhibits cardiac growth. After 4 weeks of aortic banding (transverse aortic constriction (TAC)), increases in left ventricular weight and myocyte size were significantly smaller in Tg than in NTg, indicating that GSK-3α inhibits cardiac hypertrophy. More severe cardiac dysfunction developed in Tg after TAC. Increases in fibrosis and apoptosis were greater in Tg than in NTg after TAC. Among signaling molecules screened, ERK phosphorylation was decreased in Tg. Adenovirus-mediated overexpression of GSK-3α, but not GSK-3β, inhibited ERK in cultured cardiac myocytes. Knockdown of GSK-3α increased ERK phosphorylation, an effect that was inhibited by PD98059, rottlerin, and protein kinase Cϵ (PKCϵ) inhibitor peptide, suggesting that GSK-3α inhibits ERK through PKC-MEK-dependent mechanisms. Knockdown of GSK-3α increased protein content and reduced apoptosis, effects that were abolished by PD98059, indicating that inhibition of ERK plays a major role in the modulation of cardiac growth and apoptosis by GSK-3α. In conclusion, up-regulation of GSK-3α inhibits cardiac growth and pressure overload-induced cardiac hypertrophy but increases fibrosis and apoptosis in the heart. The anti-hypertrophic and pro-apoptotic effect of GSK-3α is mediated through inhibition of ERK.

Effect of extracellular pH on sodium current in isolated, single rat ventricular cells.

SummaryEffects of extracellular pH on the sodium current (INa) of single rat ventricular cells were examined under conditions of voltage clamp and internal perfusion. In this way, pHi was controlled while pHo was changed. The combined suction pipette-microelectrode method was used. The suction pipette passed current and perfused the cells interior; the microelectrode measured membrane potential. Increasing extracellular H+ depressedINa and slowed inactivation. The current-voltage curves forINa and Slowed inactivation. The current-voltage curves forINa were shifted to positive and negative potentials at low and high pHo, respectively. Similar potential shifts were observed in both the conductance voltage curve and the steadystate inactivation voltage curve (h∞). Conduction was also depressed at low pHo. The shifts were probably due to surface charge effects, while the impaired conduction was probably due to protonation of a site in the Na channel.

Voltage‐dependent activation of potassium current in Helix neurones by endogenous cellular calcium.

1. The effect of endogenous Ca on potential‐dependent K current IKD, was examined in identifiable neurones of Helix aspersa. The suction pipette method of internal perfusion was used along with a combined voltage‐clamp method in which the membrane potential was measured by a separate glass micro‐electrode and the current was passed by the suction pipette. Activation of the potential‐dependent A current, IA, was prevented by using holding potentials of ‐40 mV where IA is inactivated and by the addition of the A‐current blocker 4‐aminopyridine. Activation of K currents by transmembrane Ca current, IKCa, was suppressed by Co substitution for Ca ion extracellularly. 2. Under these conditions, IKD rose to a peak value and then subsided to a steady level. The current‐voltage (I‐V) relationship for peak IKD had an upward bump at about +50 mV that gave it an S‐shape. The I‐V curve for steady IKD rose continuously. Peak and steady IKD were reduced by perfusing with EGTA or F ions intracellularly. The EGTA effect occurred at intracellular Ca activity levels below 10(‐7) M. Increases in the concentration of EGTAi at constant Cai had no additional effect; however, recovery experiments do not allow us to rule out some direct action of EGTA on IKD. 3. Prolonged extracellular perfusion with Co‐substituted solutions also reduced IKD and the effects occurred more quickly when the solutions were made hypertonic or caffeine was added to them. The peak transient was abolished, and the small remaining steady IKD (about 5‐10% of normal peak IKD) was blocked by tetraethylammonium. IKD could be restored by the temporary reintroduction of Ca in the extracellular solution. 4. The S‐shape of the peak I‐V relationship for IKD may be due to Ca released from an endogenous site by membrane depolarization. The reduction of steady and peak IKD to very low values by Ca chelators or prolonged perfusion with Ca‐free solutions indicates that Cai is important for activation of these K channels. 5. Three cellular structures were identified in electron micrographs of freeze‐fractured neurones that could be involved in potential‐dependent endogenous Ca release. These were a restricted extracellularly space, an intracellular membrane system of endoplasmic reticulum that may be fused to the internal face of the plasma membrane (the subsurface cisterns of Henkart & Nelson, 1979), and intracellular vesicles that also may be fused to the plasma membrane.

New Positive Inotropic Agent OPC-8212 Modulates Single Ca2+ Channels in Ventricular Myocytes of Guinea Pig

Summary: The effects of a new positive inotropic drug, OPC-8212 (a quinolinone derivative, 3,4-dihydro-6-[4-(3,4-dimethoxybenzoyl)-1-piperazinyl]-2(1H)-quinolinone), on whole-cell and single-channel calcium currents of guinea pig ventricular myocytes were studied by the patch-clamp method. OPC-8212 increased whole-cell calcium currents in a concentration-dependent manner. The concentration–response curve was fitted by a single binding site model that has an ED50 of 2.7 μM. At the single-channel level, OPC-8212 increased calcium currents mainly by increasing the probability of channel opening on depolarization; the open times were slightly increased, but single-channel conductance did not change. The actions of OPC-8212 on single calcium channel currents were compared with those of forskolin, isoproterenol, and caffeine, agents that cause positive inotropic effects associated with an increase in cytoplasmic cyclic AMP level, and with those of the dihydropyridine calcium channel agonist (−)Bay K 8644, an agent that has a positive inotropic effect but does not act through cyclic AMP. OPC-8212 increased calcium currents in a manner that resembled the actions of forskolin, isoproterenol, and caffeine rather than the actions of (−)Bay K 8644. We suggest that activation of calcium currents by OPC-8212 is mediated by an elevation of intracellular cyclic AMP. Since OPC-8212 produces bradycardia and antitachycardiac effects in animals and humans, unlike agents that increase cyclic AMP, additional effects on other currents are likely.

Blockage of the sodium current in isolated single cells from rat ventricle with mexiletine and disopyramide

The blocking effects of local anesthetics, mexiletine and disopyramide on the sodium currents (INa) of enzymatically isolated, single cells from rat ventricle were studied under voltage clamp conditions. A suction pipette technique was used for voltage clamp and internal perfusion. Potassium currents were blocked by replacing K+ with Cs+ in the internal and external solutions; calcium currents were blocked by replacing Ca2+ with Co2+ in the external solution to isolate INa. When the cells were stimulated infrequently (less than 1 Hz), both drugs produced dose-dependent depression of INa, which was correlated with one-to-one binding to sodium channel. A half-blocking concentration (KD) of 2.8 X 10(-5) M was observed for both agents. The shape of the current-voltage curve along the voltage axis remained unchanged in the presence of either drug. Both drugs shifted the inactivation curve of INa to more negative potentials. Mexiletine produced a marked use-dependent blockage of INa, whereas disopyramide did not produce significant use-dependent block under similar experimental conditions. Both drugs prolonged the recovery of INa from inactivation. The results suggested that both drugs interact with the inactivation mechanism of the sodium channels of rat myocardial cells.

Effects of a new antiarrhythmic compound SUN 1165 [N-(2,6-dimethylphenyl)-8-pyrrolizidineacetamide hydrochloride] on the sodium currents in isolated single rat ventricular cells

SummaryThe effects of a new antiarrhythmic compound, SUN 1165 (which resembles lidocaine in chemical structure) on sodium currents (INa) of enzymatically isolated, single rat ventricle cells were studied under current or voltage clamp conditions. A suction pipette technique was used for current injection and internal perfusion. Potassium currents were blocked by replacing K+ with Cs+ in the internal and external solutions, and calcium current by replacing Ca2+ with Co2+ in the external solution. When cells were stimulated infrequently (<1 Hz), SUN 1165 decreased INa without changing the position of the current-voltage curve. The inactivation curve of INa shifted to negative potentials along the voltage axis. The drug also produced an additional use-dependent block of INa, which depended on the frequency of the voltage clamp pulse. The effects of SUN 1165 on INa resemble those of lidocaine, although the relative importance of use-dependent action versus tonic blockade differs from that of lidocaine.

Molecular basis of regulation of ionic channels by G proteins

Publisher Summary This chapter presents primary structure of G proteins as deduced from purified proteins and cloned subunits. It also discusses their functions and presents data on direct regulation of ionic channels by G proteins. Experiments on expression of subunits, either in bacteria or by in vitro translation of messenger RNA (mRNA) synthesized from complimentary DNA (cDNA), are discussed as tools for definitive assignment of function to a given G protein. The chapter also discusses the dynamics of G protein-mediated signal transduction. The key points discussed in the chapter include the existence of two superimposed regulatory cycles in which G proteins dissociate into α plus βγ upon activation by guanosine triphosphate (GTP) and where the dissociated α subunits hydrolyze GTP. The chapter emphasizes the action of receptors to catalyze rather than regulate by allostery the activation of G proteins by GTP and also the role of subunit dissociation, without which receptors cannot act as catalysts. It also provides an overview on intramembrane networking of G protein-mediated receptor-effector coupling.