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Biomedical subjects

I Imanaga

Publications and source records attributed to I Imanaga.

At least 37 records · Page 2Linked to original sources
PubMed1996

Fluctuation analysis of nonselective cation currents induced by AIF complex in guinea-pig chromaffin cells.

Properties of aluminium fluoride (AIF) complex-activated nonselective cation (NS) channels in guinea-pig chromaffin cells were investigated using the patch clamp technique. As the membrane potential was hyperpolarized from the holding potential of -55 mV, the AIF-induced nonselective cation current (INS) diminished progressively. With hyperpolarizations to -100 mV or more negative potentials, the AIF.INS almost instantaneously disappeared. The apparent unit conductance of AIF INS was estimated to be 3 pS by fluctuation analysis. The open state probability of AIF-activated NS channels became large with a decrease in concentration of free Mg2+ ions inside the cell and was less than 0.5 at 12 microM Mg2+. It is concluded that NS channels in the chromaffin cell apparently differ from those in smooth muscle cells.

Aluminum Compounds↗
PubMed1996

ADP indirectly supports activation of non-selective cation channels by AlF complex in guinea-pig chromaffin cells.

Infusion of the G protein activator, AlF complex, into guinea-pig adrenal chromaffin cells produced an inward non-selective cation current (INS) at -55 mV. Under metabolically suppressed conditions, this current was abolished by removal of ATP from the AlF-containing pipette solution. ADP could substitute for ATP in generation of the current. This supporting action of ADP was eliminated by addition to the AlF solution of the ATP-consuming system (hexokinase and 2-deoxyglucose). A similar generation of INS occurred when AMP and phosphocreatine were simultaneously added to AlF solution, but not when either of the agents was added separately. These results suggest that under conditions of whole-cell current recordings, the machinery for ATP production is preserved and that ADP indirectly supports generation of INS by the AlF complex.

Adenosine Diphosphate↗
PubMed1996

Modulation of ryanodine binding to the cardiac Ca2+ release channel by arachidonic acid.

Effects of arachidonic acid (AA) on the Ca2+ release channels in cardiac sarcoplasmic reticulum were examined by the 3H-ryanodine binding method. The samples used were membrane vesicles of junction sarcoplasmic reticulum (JSR) and solubilized ryanodine receptor proteins. AA inhibited the amount of hot ryanodine bound to its receptor in both types of samples and this inhibitory effect was dose-dependent. The Khalf values of the dose-response curve were 12 and 97 microM in the JSR membrane vesicles and the solubilized proteins, respectively. Moreover, Michaelis, Scatchard and Lineweaver-Burk analyses were performed to evaluate Kd, Bmax and Kd/Bmax values. During exposure to AA, the Kd value increased while the Bmax value decreased. These results suggest that AA directly modifies the structure of the ryanodine binding site.

Animals↗
PubMed1996

The protective effect of D-sotalol against hypoxia-induced myocardial uncoupling.

The effects of D-sotalol on intercellular electrical coupling and ultrastructure under hypoxic conditions were investigated in myocardial samples from eight young (1-2 months) and four older (10-12 months) guinea pigs. A right ventricular muscle strip was kept simultaneously in two divided chambers and superfused with normoxic and/or hypoxic (97% N2+ 3% Co2) Krebs solution. Hypoxia caused shortening of action potential duration (APD) and electrical cell-to-cell uncoupling. If the uncoupling appeared after short-term hypoxia (less than 30 min), administration of 3.10(-7)M of D-sotalol to the hypoxic perfusate led to a recovery of electrical coupling. Transmission electron microscopy revealed moderate reversible ultrastructural alterations of the cardiomyocytes. No apparent changes in intercellular junctions were observed. The recoupling effect of sotalol decreased with the time of hypoxia as the ultrastructural damage progressed. After prolonged hypoxia (more than 30 min), cardiomyocytes were markedly injured, intercellular junctions were severely affected, and gap junctions occurred less frequently. In these cases, administration of D-sotalol caused only transient recoupling. After 1 h of hypoxia, no recoupling was observed. Pretreatment with D-sotalol prevented hypoxia-induced electrical uncoupling and markedly attenuated ultrastructural damage, although shortening of APD still persisted. Our results indicate that the cardioprotective effect of D-sotalol on electrical intercellular coupling is closely associated with sotalol-induced prevention of the ultrastructural damage. Considering previous results, we suggest that this protective effect of D-sotalol may be related to its ability to increase intracellular cyclic adenosine monophosphate and, thereby, to decrease cytosolic free Ca. These effects can explain the antiarrhythmic and defibrillating properties of D-sotalol.

Action Potentials↗
PubMed1996

Role and source of ATP for activation of nonselective cation channels by AlF complex in guinea pig chromaffin cells.

Intracellular dialysis with the solution containing the G protein activator, AlF complex, induced an inward nonselective cation current (INS) at -55 mV in chromaffin cells. Amplitudes of INS induced by dialysis with ATP-free AlF solutions progressively diminished as cells were pretreated with cyanide, a mitochondrial inhibitor. After a 10-min pretreatment, generation of INS by the AlF complex depended on exogenous ATP delivered from pipette solution. The relationship between amplitudes of INS and concentrations of MgATP was well expressed by a rectangular hyperbola with an EC50 of 0.265 mM. This result suggests that the cyanide treatment almost depleted ATP near the plasma membrane. On the other hand, a similar cyanide treatment of adrenal medullary preparations did not induce a marked decrease in cellular ATP content. GTP, ITP, or UTP could not substitute for ATP in generation of INS by the AIF complex. Similarly, the substitution of ATP with non- or poorly hydrolyzable ATP analogues did not aid in generating INS. Bath application of the kinase inhibitor, H-7 (100 microM), suppressed AlF-induced INS in a manner depending on intracellular Mg2+. We conclude that ATP is a prerequisite for generation of INS as a phosphoryl donor and that mitochondria is the main source of ATP.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1996

Rectification of rabbit cardiac ryanodine receptor current by endogenous polyamines.

The actions of three endogenous polyamines (spermine, spermidine, and putrescine) were defined on Ca2+ release channels (ryanodine receptors, RyRs) isolated from rabbit cardiac sarcoplasmic reticulum. The current-voltage relationship of the RyR channel was N-shaped in the presence of polyamine (1-5 mM). Polyamine blocked conduction near 0 mV, but the blockade was relieved at large potentials. Polyamines acted (blocked) from both sides of the channel. Polyamine efficacy was dependent on current direction and was inversely related to the ion selectivity of the RyR pore. This suggests that polyamine interacts with current-carrying ions in the permeation pathway. The apparent half-block concentration of spermine at 0 mV was < 0.1 mM. The features of polyamine blockade suggest that the polyamines are permeable cationic blockers of the RyR channel. Further, the levels of polyamines found in muscle cells are sufficient to block single RyR channels and thus may alter the sarcoplasmic reticulum Ca2+ release process in situ.

Animals↗
PubMed1996

Mn(2+)-induced transient contraction of the longitudinal muscle of guinea-pig stomach through prostaglandin synthesis.

1. A low concentration of Mn2+ (less than 0.3 mM) transiently enhanced a contractile force (Mn(2+)-induced TC) of the longitudinal muscles of the guinea-pig stomach. 2. The Mn(2+)-induced transient contraction (TC) was not blocked by TTX (10(-7) M) or atropine (10(-6) M), nor by nifedipine (10(-6) M) or D-600 (10(-6) M), but was blocked by Ca2+ removal from the Krebs solution. 3. A preapplication of indomethacin (10(-7) M) completely inhibited an induction of the Mn(2+)-induced TC, but exogenous PGE2 (10(-7) M) was able to induce Mn(2+)-induced TC even with the presence of indomethacin (10(-7) M) and Mn2+ (0.1 mM) in the Krebs solution. 4. Quinacrine (10(-5) M), a phospholipase A2 inhibitor, partially inhibited the Mn(2+)-induced TC. 5. These results suggest that Mn(2+)-induced TC is probably mediated through cyclooxygenase and the subsequent generation of prostaglandin leading to the contraction.

Animals↗
PubMed1995

Mg2+-dependent phosphatase as an inhibitory mediator of the nonselective cation current induced by aluminum fluoride in guinea-pig chromaffin cells.

Internal administration of the G protein activator, guanosine-5'-o-(3-thiotriphosphate) (GTP gamma S) or aluminum fluoride (AIF) complex, produced an inward nonselective cation current (INS) at -55 mV. This current was rapidly diminished under conditions of high intracellular Mg2+ ([Mg2+] = 979 microM), the half decay time (T1/2) being 80 to 100 s. As [Mg2+] in AlF solutions decreased from 400 to 12 microM, the maximum amplitude of AlF-induced INS became larger and the current was diminished more slowly. The AlF INS in the presence of 12 microM Mg2+ reversed polarity at about +9 mV, irrespective of the extent of decline. Bath application of muscarine produced a sustained INS in the absence of AlF complex, but in its presence, the overall current comprising a spontaneously developed INS and muscarine-induced INS was rapidly diminished. Addition of vanadate (0.5 mM) to 979 microM Mg2+ -containing AlF solution mimicked the effects of low Mg2+ solution. Inversely, addition of alkaline phosphatase (40 units/ml) to 12 microM Mg2+ AlF solution reproduced the effects of high Mg2+ solution. It is suggested that AlF complex deactivates INS through facilitating an apparent activity of Mg2+ -dependent phosphatase.

Aluminum Compounds↗
PubMed1995

Phosphatidylinositol hydrolysis is involved in production of Ca(2+)-dependent currents, but not non-selective cation currents, by muscarine in chromaffin cells.

Whether phosphatidylinositol hydrolysis and a subsequent Ca2+ mobilization are responsible for muscarine-induced transient outward currents (IO) and non-selective cation currents (INS) in the guinea-pig chromaffin cell was investigated using the perforated patch method. IO, but not INS, failed to be reproduced in Ca(2+)-free solution and was markedly reduced by prior exposure to caffeine under Ca(2+)-free conditions or by addition to normal solution of cyclopiazonic acid (CPA), a Ca2+ ATPase inhibitor. Application of CPA in Ca(2+)-free solution, however, suppressed INS by about 50% in 73% of the cells tested. Bath application of 1.5 mM neomycin, a phospholipase C inhibitor, induced the time-dependent decline of IO with near abolition at 20 min or less, whereas it produced a time-independent decrease of INS and an inwardly rectifying K+ current. INS in the presence or absence of neomycin was well fitted to rectangular hyperbolas with the same ED50 of 2.17 microM, but with a 33% smaller maximum amplitude in the former, indicating a non-competitive inhibition by neomycin. We conclude that, while phosphatidylinositol hydrolysis mediates the production of IO, it does not mediate that of INS by muscarine.

Animals↗
PubMed1995

Phosphatase is responsible for run down, and probably G protein-mediated inhibition of inwardly rectifying K+ currents in guinea pig chromaffin cells.

The mechanism of G protein-mediated inhibition of an inwardly rectifying K+ current (IIR) in adrenal chromaffin cells was investigated using the whole-cell version of the patch clamp technique. In case of recording with use of ATP-containing patch solution, the IIR was well maintained; otherwise, it ran down within 15 min. This run down was not prevented by replacement with adenylyl-imidodiphosphate, a nonhydrolysable analogue of ATP, but was markedly reduced by the addition to the ATP-free solution of 1 microM calyculin A, a specific inhibitor of serine/threonine phosphatase 1 (PP1) and 2A (PP2A). The addition of alkaline phosphatase to the ATP-containing solution facilitated run down of the current, and application of 100 microM H-7, a general kinase inhibitor, reversibly suppressed IIR. These results taken together suggest that inwardly rectifying K+ channels are under the influence of kinase and phosphatase without external signals. Infusion of nonhydrolysable analogues of GTP, guanosine-5'-O-(3-thiophosphate) (GTP gamma S) or guanylyl-imidodiphosphate, through the pipette produced little inward current at -55 mV, but completely inhibited IIR within approximately 5 or 6 min in all cells tested in the presence of 12 microM Mg2+ inside the cell. In contrast, infusion of aluminum fluoride (AlF) complex, another GTP binding (G) protein activator, consistently produced large inward currents, but did not alter IIR noticeably for 15 min in 17% of the cells tested. In the other cells, the inhibition of IIR developed slowly after long latent periods. This inhibitory potency of AlF was not enhanced by an increase in Mg2+ concentrations. Subtraction of the current-voltage relationship before from that noted during the generation of inward current by AlF complex revealed that the inward current diminished progressively with hyperpolarizations, as is the case with a nonselective cation current (INS) induced by a muscarinic agonist. Thus, AlF complex seems to be potent with the generation of INS, but not with IIR inhibition. The addition of 3 microM calyculin A significantly retarded the IIR inhibition by GTP gamma S, whereas that of 1 microM okadaic acid, another inhibitor of PPI and PP2A, markedly prevented the decline of IIR by AIF complex. Our observations suggest that the low potency of AlF complex in inhibiting IIR may be due to interference with phosphatase activity and that the activation of G protein suppresses IIR, probably by enhancing the apparent activity of phosphatase, which may explain run down of the current.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1995

Mechanism of activation of nonselective cation channels by putative M4 muscarinic receptor in guinea-pig chromaffin cells.

1. Mechanisms involved in the generation of nonselective cation currents (INS) by muscarinic agonists in the chromaffin cell were investigated by the perforated patch method. 2. Bath application of muscarine (0.1-30 microM) produced an inward INS with or without a transient outward current at -40 mV, whereas oxotremorine (0.06-60 microM) induced INS alone. Rectangular hyperbolas with EC50s of 2.01 and 0.21 microM were fitted to muscarine- and oxotremorine-induced INSS, respectively, and the maximal amplitude of the former was about 3.4 times larger than that of the latter. 3. In 36% of the cells exposed to Ca(2+)-free solution, muscarine INS was suppressed, being 53% of control 20 min after the perfusion, and in four cells that were incubated with Ca(2+)-free solution for 2 h or more, the INS averaged 44% of that induced subsequently in normal solution. In contrast, muscarine INS was enhanced by about 30% when A-23187 was added to normal solution. 4. W-7 and W-5, calmodulin-related agents, were almost equally potent in inhibiting muscarine INS, whereas compound 5, a potent inhibitor of calmodulin-dependent kinase II (CaM kinase II), produced no evident inhibition. 5. HA1004, a weak kinase C inhibitor, induced a reversible suppression of muscarine INS with an IC50 of 163 microM, whereas H-8, another kinase inhibitor, produced an even small degree of inhibition. Administration of phorbol 12, 13-dibutyrate did not mimic muscarinic stimulation of NS channels; rather, it led to a progressive inhibition of INS and this inhibition was almost complete within 20 min. An inactive phorbol ester had no such effect. 6. The muscarinic antagonists, pirenzepine and AF-DX 116, shifted the dose-response curve for the muscarine INs to the right in a parallel manner. The KDS for pirenzepine and AF-DX 116 were estimated to be 13 nM (95% confidence interval, 11-16 nM) and 365 nM (283-470 nM), respectively.7. These results suggest that muscarine efficiently produces INS, probably through binding to the M4 subtype, that intracellular Ca2+ has a facilitating, but not an essential role in the generation of INs, and that neither CaM kinase II nor protein kinase C is involved.

Animals↗
PubMed1994

Calcium modulation of single SR potassium channel currents in heart muscle.

The modulating effects of Ca2+ on single K+ channel currents in canine heart sarcoplasmic reticulum were studied using a planar lipid bilayer technique. The open-state probability and the unitary open-state current both decreased gradually as the Ca2+ concentration was reduced from pCa 3 to pCa 7.5. Each single-channel I-V curve was ohmic at any pCa: the modulating effect of Ca2+ within this range was voltage independent. The Ca2+ dose-response curves for the conductances and open probabilities were all biphasic in shape for both sides of the channel at the voltages used. However, Ca2+ within the pCa ranges used caused significantly more prominent activation of conductance and gating properties on the cytoplasmic side than it did on the SR luminal side. Furthermore, conductance decreased when cytoplasmic Ca2+ concentrations were greater than pCa 3. The I-V relation in this instance exhibited inward rectification caused by a voltage-dependent fast block. This suggests that cardiac SR K+ channel currents may be activated or inhibited through various types of Ca2+ binding sites on and within the channels.

Animals↗
PubMed1994

The potentiation of carbachol-induced transient contractions in guinea-pig stomach muscles by a low temperature.

A transient contraction in guinea-pig stomach muscles was induced by carbachol (CCh, 10(-6) M) in a Ca(2+)-free Krebs solution. This CCh-induced transient contraction (CITC) was remarkable from 22 to 26 degrees C, but not so at other temperatures in most preparations. When a second CCh (10(-6) M) was applied to the tissue with rinsing off CCh 5 min after an application of the first CCh for 10 min, no CITC was observed. The peak amplitude of the CITC increased with the CCh concentration dose-dependently, and that of CITC (by 10(-6) M CCh) declined exponentially over time after washing the tissue with a Ca(2+)-free Krebs solution. The decline time constant increased from 4.8 +/- 1.2 min (mean +/- SEM; n = 5) at 35 degrees C to 13.2 +/- 2.5 min (mean +/- SEM; n = 5) at 22 degrees C. Furthermore, the falling phase of CITC (by 10(-6) M CCh) also showed an exponential decay of 7.4 +/- 0.2 s (mean +/- SEM; n = 6) at 35 degrees C in the time constant. This time constant increased to 22.3 +/- 0.3 s (mean +/- SEM; n = 6) at 22 degrees C. From the above results, it is concluded that CITC may be due to the Ca(2+)-release from an intracellular store site while released Ca2+ may be immediately excluded to an extracellular space by the Ca2+ pump of the plasma membrane, but the Ca2+ pump activity may be reduced and in addition the stored Ca2+ is kept for long time by a low temperature, which thus results in an increase in the amplitude and a prolongation of the falling phase of CITC.

Animals↗
PubMed1993

Masking of A-type K+ channel in guinea pig cardiac cells by extracellular Ca2+.

Removal of extracellular Ca2+ induced transient outward currents (Io) at membrane potentials more positive than 0 mV in the guinea pig cardiac cell. This current reached a peak within a few milliseconds of stimulation, then decreased exponentially. External Cd2+ (0.1 mM) mimicked the inhibitory effect of Ca2+ on Io. Addition of D 600 (1 microM) or quinidine (0.1 mM) in the perfusate produced a reversible suppression, and replacement of internal K+ with tetraethylammonium induced a complete inhibition of Io. The steady-state inactivation of the transient component of Io was expressed by a Boltzmann relation with a half-inactivation voltage of -33.5 mV and a slope factor of 7.5 mV. This transient component was completely or almost completely inhibited by substitution of 4-aminopyridine for external cations. We conclude that in guinea pig cardiac cells, extracellular Ca2+ at physiological concentrations is masking the activity of an A-type K+ channel. This finding implies that even should a channel gene or transcript be identified using molecular biological techniques, the channel may not necessarily function under physiological conditions.

Animals↗
PubMed1993

Phosphorylation-dependent regulation of nonselective cation channels in guinea pig chromaffin cells.

In guinea pig chromaffin cells, acetylcholine induces a nonselective cation current by activating a muscarinic receptor, probably m4. We investigated activation and deactivation processes of this current. The calmodulin antagonists trifluoperazine and calmidazolium reversibly suppressed the muscarinic activation of the nonselective cation current in a dose-dependent manner; the former was about six times less potent than the latter. H-7, a kinase inhibitor, also reversibly inhibited the muscarinic current with a concentration eliciting 50% of maximal inhibition of 277 microM. Internal application of vanadate, a nonspecific phosphatase inhibitor, enhanced the muscarinic current and markedly slowed the time course of its deactivation, but the phosphatase 1 and 2A inhibitors okadaic acid and calyculin A had no effects. Lowering Mg2+ concentration in the patch solution mimicked the effects of vanadate on the muscarinic current. Internal dialysis with vanadate or low-Mg2+ solution gradually led to development of an inward current, and the related voltage dependence was similar to that seen with the muscarinic cation current. These results suggest that kinase and Mg(2+)-dependent phosphatase are responsible for activation and deactivation of nonselective cation channels. The channels seem to be under the influence of both enzymes, even in the absence of external signals.

Animals↗
PubMed1993

G protein-mediated inhibition of inwardly rectifying K+ channels in guinea pig chromaffin cells.

Properties of inwardly directed rectification and its G protein-mediated inhibition in guinea pig chromaffin cells were studied using the whole cell version of the patch-clamp technique. The current-voltage (I-V) relationship for plateau currents in response to a 50-ms pulse showed an inwardly directed rectification between -80 and -140 mV and a negative slope at more negative potentials in normal solution. Replacement of Na+ with N-methyl-D-glucamine (NMDG) in the perfusate did not alter the plateau I-V relationship between -110 and -130 mV but did abolish the negative slope below -140 mV. The zero current or resting membrane potential in the NMDG solution was in fair agreement with the equilibrium potential for K+. The chord conductance-voltage relationship showed a good fit with the Boltzmann equation and shifted along the voltage axis by an approximate change in driving force on K+ when K+ concentration was increased. External Cs+ and Ba2+ produced a voltage-dependent inhibition of the inwardly directed rectification. These results indicate that inwardly rectifying (IR) K+ channels are mediating an inwardly directed rectification. Intracellular dialysis with guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) produced a complete suppression of this IR K+ channel, irrespective of treatment with pertussis toxin. Adding GTP or guanosine 5'-O-(2-thiodiphosphate) to the patch solution resulted in a decrease in GTP gamma S inhibition of the K+ current. Internal application of vanadate was without effect. Time course of the inhibition of the IR K+ current coincided in part with that of inactivation of a nonselective cation current. In conclusion, IR K+ channels in the chromaffin cell are subject to G protein-mediated inhibition.

Animals↗
PubMed1991

Activation by intracellular calcium of a potassium channel in cardiac sarcoplasmic reticulum.

The effects of low (pCa 7.5 to 3) concentrations of intracellular calcium ion on a single potassium channel in the sarcoplasmic reticulum of canine heart ventricular muscle were investigated using a planar lipid bilayer technique. The low concentrations were obtained by mixing EGTA and calcium chloride. By varying the pCa of the cytoplasmic face between 3 to 7.5, two novel effects were observed. First, an increase in the intracellular Ca2+ concentration produced an increase in the unit current amplitude of open states; the voltage-current relationship was ohmic at these concentrations. Second, an increase in the Ca2+ concentration increased the open probability. Both these effects of Ca2+ were dose-dependent, and were consistently observed in all channels tested. Thus, the SR potassium channel observed appears to belong to the class of Ca2(+)-activated potassium channels.

Animals↗