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Biomedical subjects

A Yatani

Publications and source records attributed to A Yatani.

At least 109 records · Page 6Linked to original sources
PubMed1987

Direct activation of mammalian atrial muscarinic potassium channels by GTP regulatory protein Gk.

The mammalian heart rate is regulated by the vagus nerve, which acts via muscarinic acetylcholine receptors to cause hyperpolarization of atrial pacemaker cells. The hyperpolarization is produced by the opening of potassium channels and involves an intermediary guanosine triphosphate-binding regulatory (G) protein. Potassium channels in isolated, inside-out patches of membranes from atrial cells now are shown to be activated by a purified pertussis toxin-sensitive G protein of subunit composition alpha beta gamma, with an alpha subunit of 40,000 daltons. Thus, mammalian atrial muscarinic potassium channels are activated directly by a G protein, not indirectly through a cascade of intermediary events. The G protein regulating these channels is identified as a potent Gk; it is active at 0.2 to 1 pM. Thus, proteins other than enzymes can be under control of receptor coupling G proteins.

Acetylcholine↗
PubMed1987

The properties of batrachotoxin-modified cardiac Na channels, including state-dependent block by tetrodotoxin.

Batrachotoxin (BTX) modification and tetrodotoxin (TTX) block of BTX-modified Na channels were studied in single cardiac cells of neonatal rats using the whole-cell patch-clamp recording technique. The properties of BTX-modified Na channels in heart are qualitatively similar to those in nerve. However, quantitative differences do exist between the modified channels of these two tissues. In the heart, the shift of the conductance-voltage curve for the modified channel was less pronounced, the maximal activation rate constant, (tau m)max, of modified channels was considerably slower, and the slow inactivation of the BTX-modified cardiac Na channels was only partially abolished. TTX blocked BTX-modified mammalian cardiac Na channels and the block decreased over the potential range of -80 to -40 mV. The apparent dissociation constant of TTX changed from 0.23 microM at -50 mV to 0.69 microM at 0 mV. No further reduction of block was observed at potentials greater than -40 mV. This is the potential range over which gating from closed to open states occurred. These results were explained by assuming that TTX has a higher affinity for closed BTX-modified channels than for open modified channels. Hence, the TTX-binding rate constants are considered to be state dependent rather than voltage dependent. This differs from the voltage dependence of TTX block reported for BTX-modified Na channels from membrane vesicles incorporated into lipid bilayers and from amphibian node of Ranvier.

Animals↗
PubMed1987

Whole-cell and single-channel calcium currents of isolated smooth muscle cells from saphenous vein.

Whole-cell and single-channel calcium currents in enzymatically isolated dog saphenous vein cells were recorded by the patch-clamp method. Test pulses to negative potentials from holding potentials of -90 to -40 mV elicited currents that inactivated quickly and in a voltage-dependent manner (called I low for low threshold). A second calcium current persisted even at relatively positive holding potentials of -30 to -10 mV, required stronger depolarizations for maximum current, and inactivated slowly (I high for high threshold). I high transported barium more than calcium, whereas I low transported the two ions equally. Single-channel current records (90 mM barium) showed a larger conductance that activated at relatively positive potentials and a smaller (about one-third) conductance that activated at weak depolarizations. Nitrendipine suppressed I high, and the effect was voltage dependent as observed in cardiac cells, although block of resting channels was much greater in vein cells (KR approximately 10(-8) M). Exposure to the stereoisomer (-)Bay K 8644 increased I high but not I low. The (-)Bay K 8644 also increased the channel activity and prolonged the open time of the larger conductance current. Thus, two types of calcium channels, differing in potential-dependence of activation and inactivation, calcium/barium selectivity, single-channel conductance, and sensitivities to dihydropyridines were identified in smooth muscle cells isolated from a large cutaneous vein.

Animals↗
PubMed1987

Reconstitution of somatostatin and muscarinic receptor mediated stimulation of K+ channels by isolated GK protein in clonal rat anterior pituitary cell membranes.

Somatostatin (SS) inhibits secretion from many cells, including clonal GH3 pituitary cells, by a complex mechanism that involves a pertussis toxin (PTX)-sensitive step and is not limited to its cAMP lowering effect, since secretion induced by cAMP analogs and K+ depolarization are also inhibited. SS also causes membrane hyperpolarization which may lead to decreases in intracellular Ca2+ need for secretion. Using patch clamp techniques we now demonstrate: 1) that both (SS) and acetylcholine applied through the patch pipette to the extracellular face of a patch activate a 55-picosiemens K+ channel without using a soluble second messenger; 2) that, after patch excision, the active state of the ligand-stimulated channel is dependent on GTP in the bath, is abolished by treatment of the cytoplasmic face of the patch with activated PTX and NAD+, and after inactivation by PTX, is restored in a GTP-dependent manner by addition of a nonactivated human erythrocyte PTX-sensitive G protein, and 3) that the 55-picosiemens K+ channel can also be activated in a ligand-independent manner with guanosine [gamma-thio] triphosphate (GTP gamma S) or with Mg2+/GTP gamma S-activated erythrocyte G protein. We call this protein GK. It is an alpha-beta-gamma trimer of which we have previously shown that the alpha-subunit is the substrate for PTX and that it dissociates on activation with Mg2+/GTP gamma S into alpha-GTP gamma S plus beta-gamma. A similarly activated and dissociated preparation of GS, the stimulatory regulatory component of adenylyl cyclase, having a different alpha-subunit but the same beta-gamma-dimer, was unable to cause K+ opening.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

A comparison between the binding and electrophysiological effects of dihydropyridines on cardiac membranes.

To investigate the mechanism of action of dihydropyridines on calcium channels, both receptor binding and the electrophysiological effects of optically pure enantiomers of Bay k 8644, Sandoz 202-791, nimodipine, and PN200-110 were studied in a guinea pig ventricular preparation. The radioligand binding studies are consistent with other reports that the inhibitory and excitatory dihydropyridines share a single binding site. The patch clamp method was used for recording whole cell calcium currents. (-)-Bay k 8644 and (+)-202-791 produced voltage-dependent increases in the calcium currents. The magnitude of the effect was dependent on the holding potential. At holding potentials between -40 and -90 mV these activators increased calcium currents in a concentration-dependent manner with EC50 values of 25 nM and 80 nM, respectively. The inhibitors (+)-Bay k 8644, (-)-202-791, (+)- and (-)-nimodipine, and (+)-PN200-110 blocked the calcium currents with potencies that depended upon holding potential. The IC50 values for these enantiomers measured at a holding potential of -80 mV were, respectively, 8000, 200, 2000, 450, and 400 nM, and IC50 values measured at a holding potential of -30 mV were 26, 1.0, 52, 4.0, and 4.5 nM. The dissociation constants calculated for some dihydropyridines are similar to the Kd values determined by radioligand binding. However, for other dihydropyridines, large discrepancies between the concentrations giving rise to half-maximal electrophysiological effects and the Kd values from binding studies could not be reconciled by voltage-dependent binding alone. We suggest that each dihydropyridine also produces unique effects on the voltage-dependent gating of calcium channels.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1987

Neurotoxins that act selectively on voltage-dependent cardiac calcium channels.

We searched for and found a toxin that acts specifically on cardiac calcium channels. This toxin, which we have designated TaiCatoxin (TCX), was purified from the venom of the Australian Taipan snake (Oxyuranus s. scutellatus), one of the world's most poisonous snakes. TCX is a highly charged, basic polypeptide with a molecular weight of 8,000. It blocks high- but not low-threshold cardiac calcium channels in a voltage-dependent manner and has no effect on potassium or sodium channels. The block occurs at nanomolar concentrations, is reversible, and is due to binding at an extracellularly facing site on the channel itself.

Animals↗
PubMed1986

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)

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1986

Diphenylhydantoin blocks cardiac calcium channels and binds to the dihydropyridine receptor.

Diphenylhydantoin was studied for its effects on Ca currents in single isolated guinea pig ventricular cells. The whole-cell patch-clamp technique was used, and Ca currents were studied after suppressing Na and K currents. At low frequencies (0.1 Hz) and negative holding potentials (-50 mV), diphenylhydantoin produced a concentration-dependent decrease in Ca currents without any significant change in the current-voltage relations. Half blocking effect occurred at 2 X 10(-4) M. The effects of diphenylhydantoin on Ca currents were dependent upon the holding potential. Inactivation curves for Ca currents were shifted to more negative potentials by the drug. The recovery of Ca currents from inactivation was prolonged by diphenylhydantoin, and the repriming of the current displayed an additional component, attributed to slow release of the drug from the channels. The voltage-dependent block was attributed to preferred binding by the inactivated channel state. Diphenylhydantoin also blocked specific [3H]-nitrendipine binding to guinea pig ventricular membrane preparations. The inhibition of [3H]-nitrendipine binding by diphenylhydantoin was competitive. Diphenylhydantoin also blocks cardiac Na channels in a voltage-dependent manner. We suggest that diphenylhydantoin binding sites exist on both Ca and Na channels.

Animals↗
PubMed1986

Reduction in the myocardial sodium current by halothane and thiamylal.

Effects of two general anesthetics, halothane and thiamylal, on the fast sodium inward current (INa) of enzymatically isolated single rat ventricular cells were studied under current clamp and voltage clamp conditions. A suction pipette technique was used for potential measurement, current injection and internal perfusion of isolated cells. In current clamp experiments, sodium action potential was elicited in a Ca-free Co Krebs solution and the action potential was reduced by 0.5% halothane and 5 X 10(-5) M thiamylal. In voltage clamp experiments, the calcium current was suppressed by replacing Ca with Co and the potassium current was eliminated by replacing K with Cs and adding 4-aminopyridine and tetraethylammonium. Both anesthetics decreased INa, in a dose dependent manner, without changing the shape of the current-voltage curve. Halothane (1%) shifted the steady state inactivation curve in a negative direction along the potential axis by 8.5 +/- 2 mV (mean +/- S.D., n = 4). Thiamylal, 5 X 10(-5) and 10(-4) M, shifted the curve in a negative direction by 4.4 +/- 0.8 mV (n = 5) and 8.6 +/- 3.2 mV (n = 5), respectively. Both agents slightly reduced the maximum sodium conductance (gNa). Halothane (1%) increased half recovery time from inactivation measured at -80 mV from 30 +/- 15 to 80 +/- 25 ms (n = 4). Thiamylal (10(-4) M) prolonged it at - 75 mV from 50 +/- 20 to 110 +/- 15 ms (n = 5). With a test pulse duration of 50 ms, neither drug produced a use-dependent inhibition of INa. Halothane and thiamylal depress the INa of cardiac muscles mainly by shifting the steady state inactivation curve in a negative direction along the potential axis. Relatively small prolongation of half recovery time from inactivation and no sign of use-dependent inhibition suggest a molecular mechanism which differs in some respects from the local anesthetics.

Animals↗
PubMed1986

Bepridil block of cardiac calcium and sodium channels.

The effects of bepridil, a new Ca channel blocking agent with reported antiarrhythmic action, were examined in single isolated ventricular cells using whole-cell patch clamp techniques. Ca currents were studied in guinea-pig ventricular cells and Na currents were studied in cultured ventricular cells from neonatal rat, a preparation which is more suitable for Na current measurements than guinea pig. At low frequencies (0.1 Hz) and negative holding potentials (-50 mV for Ca currents and -100 mV for Na currents), bepridil produced a concentration-dependent decrease in both Ca and Na currents without any significant change in the current-voltage relations. Concentration-response curves for block of Ca and Na channels were fitted by a one-to-one drug-receptor occupancy model. Half-blocking concentrations (IC50) of bepridil were 5 x 10(-7) M for Ca currents and 3 X 10(-5) M for Na currents. Bepridil had no effect on the inwardly rectifying K current and the time-dependent outward current. The effects of bepridil on Ca and Na currents depend upon the holding potential. Inactivation curves of the Ca and Na currents were shifted to more negative potentials by the drug. The recovery of both the Ca and Na currents from inactivation was always prolonged by bepridil and the repriming of both currents usually displayed an added exponential component, attributed to slow release of the drug from the channels. The results indicate that bepridil, by inhibiting both Ca and Na currents, may have clinical usefulness in the treatment of certain ischemia-induced ventricular arrhythmias.

Action Potentials↗
PubMed1985

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 ED50's 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 IC50's 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.

Animals↗
PubMed1985

Atrotoxin: a specific agonist for calcium currents in heart.

A specific label for voltage-dependent calcium channels is essential for the isolation and purification of the membrane protein that constitutes the calcium channel and for a better understanding of its function. A fraction of Crotalus atrox that increases voltage-dependent calcium currents in single, dispersed guinea pig ventricular cells was isolated. In the doses used, neither sodium nor potassium currents were changed. The fraction was active in the absence of detectable phospholipase or protease activity, and the active component, designated atrotoxin, produced its effect rapidly and reversibly. The effect was produced by extracellular but not intracellular application of the agent. The increase in Ca2+ current was blocked by the Ca2+ channel blockers cobalt and nitrendipine. The active fraction completely blocked specific [3H]nitrendipine binding to guinea pig ventricular membrane preparations. The inhibition of nitrendipine binding by atrotoxin was apparently via an allosteric mechanism. Thus atrotoxin was shown to bind to the Ca2+ channel and to act as a specific Ca2+ channel agonist.

Action Potentials↗
PubMed1985

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.

Action Potentials↗
PubMed1985

Reduction of the slow inward current of isolated rat ventricular cells by thiamylal and halothane.

The barbiturates and halothane exert a negative inotropic effect on the myocardium. A reduction in the slow inward current, carried mainly by calcium ions, is an important factor for the underlying mechanism because the calcium current during the action potential provides the calcium ions for accompanying contraction, supplies Ca ions to the sarcoplasmic reticulum for subsequent contractions, and induces Ca release from the store site. It has been suggested that reduction in the slow inward current caused by anesthetics is indicated by depression of the slow action potential of the partially depolarized myocardium. In order to assess directly the effect of anesthetics on the slow inward current, we carried out voltage clamp experiments with single isolated rat ventricular cells obtained by an enzymatic dissociation method. Thiamylal (10(-4) mol . l-1) and halothane (1%) decreased the slow inward current to 60 +/- 5% (mean +/- s.d., n = 8) and to 65 +/- 10% (mean +/- s.d., n = 8) of the control value, respectively, without changing the configuration of the current-voltage curve. The results provide further evidence for anesthetic reduction of the slow inward current of the myocardium, and suggest that the negative inotropic effect is at least partly due to the reduction in that current.

Action Potentials↗
PubMed1985

The calcium channel blocker nitrendipine blocks sodium channels in neonatal rat cardiac myocytes.

The dihydropyridine calcium channel blocker, nitrendipine, was studied for its effects on the sodium current of single cultured ventricular cells from neonatal rats. The patch-clamp method of recording whole cell currents was used, and sodium currents were isolated by suppressing potassium and calcium currents. Potassium currents were blocked by replacing potassium with cesium in the internal and external solutions and by adding tetraethylammonium chloride and 4-aminopyridine in the external solution; calcium current was blocked by replacing calcium with cobalt in the external solution. At low frequencies (0.1 Hz), nitrendipine reduced sodium currents without any significant change in the current-voltage relation. The block was dose dependent, and assuming a single occupancy model with complete block, had a half-maximum value of 3 X 10(-6) M at a holding potential of -80 mV where half the sodium channels are activatable. This value is within the range of the Kd's that have been reported for low-affinity dihydropyridine-binding sites found in cardiac sarcolemmal vesicles. In the presence of nitrendipine, the inactivation curve was shifted to hyperpolarized potentials. The block was greater with pulse intervals shorter than 1000 msec, and repriming was prolonged in the presence of the drug. These effects are similar to those of local anesthetics of the tertiary amine class, such as lidocaine. The block was relieved by the dihydropyridine agonist Bay K8644. The results are interpreted as indicating that dihydropyridines react with sodium channels.

Animals↗
PubMed1984

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.

The 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 (less than 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.

Action Potentials↗
PubMed1984

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

Effects 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 cell's interior; the microelectrode measured membrane potential. Increasing extracellular H+ depressed INa and slowed inactivation. The current-voltage curves for INa 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 steady-state inactivation voltage curve (h infinity). 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.

Animals↗