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1.
Ann Hum Genet ; 69(Pt 4): 413-28, 2005 Jul.
Article in English | MEDLINE | ID: mdl-15996170

ABSTRACT

Genetic variations in cardiac ion channels have been implicated not only as the causes of inherited arrhythmic syndromes, but also as genetic risk factors for some acquired arrhythmias. To elucidate the potential roles of genetic polymorphisms of the alpha subunit of the voltage-gated sodium channel type V (SCN5A) in cardiac rhythm disturbance, the entire SCN5A coding exons and their flanking introns were sequenced in 166 Japanese arrhythmic patients and 232 healthy controls. We detected 69 genetic variations, including 54 novel ones. Out of the 12 novel nonsynonymous single nucleotide polymorphisms (SNPs), p.Leu1988Arg was found at a frequency of 0.015. The other 11 SNPs were rare (0.001), with 6 found in arrhythmic patients and 5 in healthy controls. The frequency of a novel intronic SNP, c.703+130G>A, was significantly higher in the patients than in the controls, suggesting this SNP is associated with an unknown risk factor for arrhythmia. Following linkage disequilibrium analysis, the haplotype structure of SCN5A was inferred using high-frequency SNPs. The frequency of the haplotype harbouring both p.Leu1988Arg and the common SNP p.His558Arg (haplotype GG) was significantly lower in the patients than in the controls. This finding suggests that this haplotype (GG) might have been positively selected in the controls because of its protective effect against arrhythmias. This study provides fundamental information necessary to elucidate the effect of genetic variations in SCN5A on channel function and cardiac rhythm in Japanese, and probably in the Asian population.


Subject(s)
Arrhythmias, Cardiac/genetics , Genetic Predisposition to Disease , Haplotypes , Sodium Channels/genetics , Exons , Genetic Variation , Humans , Introns , Japan , Linkage Disequilibrium , Myocardium/metabolism , NAV1.5 Voltage-Gated Sodium Channel , Polymorphism, Genetic , Protein Structure, Secondary , Sodium Channels/chemistry , Sodium Channels/metabolism
2.
FEBS Lett ; 499(3): 256-61, 2001 Jun 22.
Article in English | MEDLINE | ID: mdl-11423127

ABSTRACT

Three novel peptides were isolated from the venomous saliva of predatory reduviids. They were identified by mass spectrometry and HPLC analysis and consist of 34-36 amino acid residues. They are relatively homologous to the calcium channel blockers omega-conotoxins from marine cone snails and belong to the four-loop Cys scaffold structural class. Ptu1, the shortest peptide, was chemically synthesized (sPtu1) and co-eluted with its native form. Circular dichroism spectra of the sPtu1 showed a high content of beta-turns similar to that of omega-conotoxins GVIA and MVIIA. Electrophysiological experiments demonstrated that sPtu1 reversibly blocks the N-type calcium channels expressed in BHK cells.


Subject(s)
Peptides/isolation & purification , Reduviidae/chemistry , Venoms/chemistry , Amino Acid Sequence , Animals , Chromatography, Liquid , Mass Spectrometry , Molecular Sequence Data , Peptides/chemistry , Protein Conformation , Sequence Homology, Amino Acid , omega-Conotoxins/chemistry
4.
FEBS Lett ; 489(1): 87-91, 2001 Jan 26.
Article in English | MEDLINE | ID: mdl-11231019

ABSTRACT

Cardiac L-type Ca(2+) channel is facilitated by protein kinase A (PKA)-mediated phosphorylation. Here, we investigated the role of Ser(1901), a putative phosphorylation site in the carboxy-terminal of rat brain type-II alpha(1C) subunit (rbCII), in the PKA-mediated regulation. Forskolin (3 microM) enhanced Ca(2+) channel currents (I(Ca)) and shifted the activation curve to negative voltages, which were abolished by protein kinase inhibitor. Replacement of Ser(1901) of rbCII by Ala abolished the enhancement of I(Ca) by forskolin but not the shift of the activation curve. These results indicate that Ser(1901) is required for the PKA-mediated enhancement of I(Ca), and that the voltage-dependence of the activation of I(Ca) appears to be modulated via another PKA phosphorylation site.


Subject(s)
Brain/metabolism , Calcium Channels, L-Type/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Serine/metabolism , Amino Acid Sequence , Animals , Cells, Cultured , Cricetinae , Cyclic AMP-Dependent Protein Kinase Type II , Molecular Sequence Data , Phosphorylation , Protein Conformation , Rabbits , Rats , Sequence Homology, Amino Acid , Serine/genetics , Transfection
5.
J Biol Chem ; 275(52): 41504-11, 2000 Dec 29.
Article in English | MEDLINE | ID: mdl-11022040

ABSTRACT

The dihydropyridine (DHP)-binding site has been identified within L-type Ca(2+) channel alpha(1C) subunit. However, the molecular mechanism underlying modulation of Ca(2+) channel gating by DHPs has not been clarified. To search for novel determinants of high affinity DHP binding, we introduced point mutations in the rat brain Ca(2+) channel alpha(1C) subunit (rbCII or Ca(v)1.2c) based on the comparison of amino acid sequences between rbCII and the ascidian L-type Ca(2+) channel alpha(1) subunit, which is insensitive to DHPs. The alpha(1C) mutants (S1115A, S1146A, and A1420S) and rbCII were transiently expressed in BHK6 cells with beta(1a) and alpha(2)/delta subunits. The mutation did not affect the electrophysiological properties of the Ca(2+) channel, or the voltage- and concentration-dependent block of Ca(2+) channel currents produced by diltiazem and verapamil. However, the S1115A channel was significantly less sensitive to DHP antagonists. Interestingly, in the S1115A channel, DHP agonists failed to enhance whole-cell Ca(2+) channel currents and the prolongation of mean open time, as well as the increment of NP(o). Responsiveness to the non-DHP agonist FPL-64176 was also markedly reduced in the S1115A channel. When S1115 was replaced by other amino acids (S1115D, S1115T, or S1115V), only S1115T was slightly sensitive to S-(-)-Bay K 8644. These results indicate that the hydroxyl group of Ser(1115) in IIIS5-S6 linker of the L-type Ca(2+) channel alpha(1C) subunit plays a critical role in DHP binding and in the action of DHP Ca(2+) channel agonists.


Subject(s)
Calcium Channels, L-Type/chemistry , Dihydropyridines/agonists , Amino Acid Sequence , Animals , Calcium Channel Blockers/pharmacology , Calcium Channels, L-Type/physiology , Cricetinae , Molecular Sequence Data , Point Mutation , Protein Conformation , Protein Subunits , Rats , Serine , Structure-Activity Relationship
6.
Eur J Pharmacol ; 404(1-2): 231-8, 2000 Sep 15.
Article in English | MEDLINE | ID: mdl-10980283

ABSTRACT

Detrusor muscle relaxes upon activation of beta-adrenoceptors on smooth muscle cells. However, the mechanism of relaxation following the stimulation of beta-adrenoceptors remains unclear. In order to clarify the mechanism, we investigated the involvement of ion channels in bladder relaxation. In guinea-pig isolated bladder strips precontracted by high-K(+), isoproterenol caused concentration-dependent relaxation. The relaxation caused by isoproterenol (1 microM) was larger in 30 mM K(+) than in 120 mM K(+) (54.2+/-8.0% and 18.2+/-4.1% of papaverine-induced relaxation, respectively, n=4). Iberiotoxin (100 nM) inhibited the isoproterenol-induced relaxation (vehicle 69.5+/-8.0% vs. iberiotoxin 24.9+/-6.2%, respectively, n=5). Whole-cell patch-clamp recording revealed that isoproterenol as well as forskolin increased the iberiotoxin-sensitive K(+) currents, and this increase was abolished by protein kinase inhibitor. These results suggest that the isoproterenol-induced relaxation of guinea-pig bladder smooth muscle is mainly mediated by facilitation of BK(Ca) channels subsequent to the activation of the cAMP/protein kinase A pathway.


Subject(s)
Muscle Relaxation/physiology , Muscle, Smooth/physiology , Potassium Channels, Calcium-Activated , Potassium Channels/physiology , Receptors, Adrenergic, beta/physiology , Animals , Guinea Pigs , In Vitro Techniques , Large-Conductance Calcium-Activated Potassium Channels , Male , Membrane Potentials/physiology , Muscle, Smooth/cytology , Urinary Bladder/cytology , Urinary Bladder/physiology
7.
Am J Physiol ; 276(4): H1178-89, 1999 04.
Article in English | MEDLINE | ID: mdl-10199841

ABSTRACT

The amplification factor of dihydropyridine (DHP)/ryanodine receptors was defined as the amount of Ca2+ released from the sarcoplasmic reticulum (SR) relative to the influx of Ca2+ through L-type Ca2+ channels in rat ventricular myocytes. The amplification factor showed steep voltage dependence at potentials negative to -10 mV but was less dependent on voltage at potentials positive to this value. In cells dialyzed with 0.2 mM cAMP in addition to 2 mM fura 2, the Ca2+-channel agonist (-)-BAY K 8644 enhanced Ca2+-channel current (ICa), shifted the activation curve by -10 mV, and significantly delayed its inactivation. Surprisingly, BAY K 8644 reduced the amplification factor by 50% at all potentials, even though the caffeine-releasable Ca2+ stores were mostly intact at holding potentials of -90 mV. In contrast, brief elevation of extracellular Ca2+ activity from 2 to 10 mM enhanced both ICa and intracellular Ca2+ transients in the absence or presence of BAY K 8644 but had no significant effect on the amplification factor. BAY K 8644 abolished the direct dependence of the rate of inactivation of ICa on the release of Ca2+ from the SR. These findings suggest that the gain of the Ca2+-induced Ca2+ release in cardiac myocytes is regulated by the gating kinetics of cardiac L-type Ca2+ channels via local exchange of Ca2+ signals between DHP and ryanodine receptors and that BAY K 8644 suppresses the amplification factor through attenuation of the Ca2+-dependent inactivation of Ca2+ channels.


Subject(s)
3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester/pharmacology , Calcium Channel Agonists/pharmacology , Calcium Channels/physiology , Calcium Signaling/drug effects , Myocardium/metabolism , Ryanodine Receptor Calcium Release Channel/physiology , Animals , Calcium/metabolism , Calcium Channels/drug effects , Calcium Channels, L-Type , Electric Conductivity , Extracellular Space/metabolism , Heart Ventricles , Myocardium/cytology , Osmolar Concentration , Rats , Sarcoplasmic Reticulum/drug effects , Sarcoplasmic Reticulum/metabolism
8.
Eur J Pharmacol ; 358(3): 277-87, 1998 Oct 09.
Article in English | MEDLINE | ID: mdl-9822895

ABSTRACT

We have previously shown, in a radioligand binding study with single ventricular myocytes, that benzothiazepine and dihydropyridine binding sites interact with each other. To further examine whether this interaction between the two binding sites is reflected in the function of L-type Ca2+ channels, the blocking action of diltiazem, nitrendipine, and the combination of these two drugs on L-type Ca2+ channel currents was investigated using baby hamster kidney cells expressing the alpha 1C, alpha 2/delta, beta and gamma subunits of the Ca2+ channel. The effects of diltiazem and nitrendipine were additive at room temperature but synergistic at 33 degrees C. The use-dependent block by 3 microM of diltiazem was significantly enhanced from 28% to 68% by addition of 30 nM of nitrendipine, which by itself did not have a blocking effect. Thus, we conclude that benzothiazepine- and dihydropyridine binding sites interact and potentiate their blocking action on L-type Ca2+ channels in a temperature-dependent manner.


Subject(s)
Calcium Channel Blockers/pharmacology , Calcium Channels/drug effects , Dihydropyridines/pharmacology , Thiazepines/pharmacology , Animals , Binding Sites , Binding, Competitive , Calcium Channel Blockers/metabolism , Calcium Channels/metabolism , Cell Line , Dihydropyridines/metabolism , Diltiazem/pharmacology , Membrane Potentials/drug effects , Myocardium/cytology , Myocardium/metabolism , Nitrendipine/pharmacology , Patch-Clamp Techniques , Temperature , Thiazepines/metabolism , Time Factors
9.
J Gen Physiol ; 109(6): 717-29, 1997 Jun.
Article in English | MEDLINE | ID: mdl-9222898

ABSTRACT

We have produced transgenic mice which overexpress cardiac Na(+)-Ca2+ exchange activity. Overexpression has been assessed by Western blot, Northern blot, and immunofluorescence. Functional overexpression was analyzed using membrane vesicles and isolated ventricular myocytes. In whole cell clamped myocytes dialyzed with 0.1-0.2 mM Fura-2, the magnitude of ICa and Ca2+i-transient triggered by ICa or caffeine were not significantly different in transgenic vs. control myocytes. In transgenic myocytes, activation of ICa, however, was followed by a large slowly inactivating transient inward current representing INa-Ca. This current depended on Ca2+ release as it was abolished when sarcoplasmic reticulum (SR) Ca2+ was depleted using thapsigargin. Cai-transients triggered by rapid application of 5 mM caffeine, even though equivalent in control and transgenic myocytes, activated larger INa-Ca (approximately 5 pA/pF at -90 mV) in transgenic vs. control myocytes (1.5 pA/pF). The decay rate of caffeine-induced Ca2+i-transient and INa-Ca was 2.5 times faster in transgenic than in control myocytes. 5 mM Ni2+ was equally effective in blocking INa-Ca in control or transgenic myocytes. In 9 out of 26 transgenic myocytes, but none of the controls, Ca2+ influx via the exchanger measured at +80 mV caused a slow rise in [Ca2+]i triggering rapid release of Ca2+ from the SR, SR Ca2+ release triggered by the exchanger at such potentials was accompanied by activation of transient current in the inward direction. In 2 mM Fura-2-dialyzed transgenic myocytes caffeine-triggered Cai-transients failed to activate INa-Ca even though the kinetics of inactivation of ICa slowed significantly in caffeine-treated myocytes. In 0.1 mM Fura-2-dialyzed transgenic myocytes 100 microM Cd2+ effectively blocked ICa and suppressed Cai-transients at -10 or +50 mV. Our data suggests that in myocytes overexpressing the exchanger, the content of intracellular Ca2+ pools and the signaling of its release by the Ca2+ channel vis-à-vis the Na(+)-Ca2+ exchanger were not significantly altered despite an up to ninefold increase in the exchanger activity. We conclude that the exchanger remains functionally excluded from the Ca2+ microdomains surrounding the DHP/ryanodine receptor complex.


Subject(s)
Calcium/metabolism , Gene Expression/genetics , Ion Transport/physiology , Myocardium/metabolism , Sodium/metabolism , Animals , Immunohistochemistry , Mice , Mice, Transgenic , Patch-Clamp Techniques
10.
Eur J Pharmacol ; 325(2-3): 229-36, 1997 May 01.
Article in English | MEDLINE | ID: mdl-9163570

ABSTRACT

The effects of a 1,5-benzothiazepine derivative, (+)-cis-3-(acetyloxy)-5-[2-[[2-(3,4-dimethoxyphenyl)ethyl]-methyla mino]ethyl]-2,3-dihydro-2-(4-methyoxyphenyl)-1,5-benzothiazepine-4 (5H)-one (DTZ323), on membrane currents were investigated in guinea-pig ventricular myocytes using the whole-cell patch-clamp technique. DTZ323 suppressed the L-type Ca2+ channel currents (I[Ca(L)]) more selectively than the T-type Ca2+ channel and the Na+ channel currents. DTZ323 inhibited I[Ca(L)] in a use- and a voltage-dependent manner with 24 times higher potency than that of diltiazem. Rate of recovery of I[Ca(L)] from the conditioned block by DTZ323 was faster compared with diltiazem and verapamil, and was steeply dependent on the holding potential at resting membrane potential range in ventricular myocytes (-90 to -60 mV). Our results suggest that DTZ323 is a selective Ca2+ channel antagonist, the most potent among the 1,5-benzothiazepine Ca2+ channel antagonists, and that the voltage- and use-dependent effect of DTZ323 on I[Ca(L)] is due to the steep voltage dependence of the rate of dissociation from the cardiac L-type Ca2+ channels.


Subject(s)
Calcium Channel Blockers/pharmacology , Diltiazem/analogs & derivatives , Heart/drug effects , Myocardium/metabolism , Animals , Calcium Channels/classification , Calcium Channels/drug effects , Calcium Channels/metabolism , Diltiazem/pharmacology , Guinea Pigs , Heart Ventricles/cytology , Heart Ventricles/drug effects , Heart Ventricles/metabolism , In Vitro Techniques , Kinetics , Male , Membrane Potentials/drug effects , Myocardium/cytology , Patch-Clamp Techniques , Sodium Channels/drug effects , Sodium Channels/metabolism , Verapamil/pharmacology
11.
J Pharmacol Exp Ther ; 281(1): 173-9, 1997 Apr.
Article in English | MEDLINE | ID: mdl-9103495

ABSTRACT

A novel derivative of diltiazem (1,5-benzothiazepine Ca++ antagonist), DTZ323, 3-(acetyloxy)-5-[2-[[2-(3,4-dimethoxyphenyl)ethyl]-methylamino]eth yl]-2,3-dihydro-2-(4-methoxyphenyl)-1,5-benzothiazepine-4(5H)-one, was characterized by radioligand binding experiments with rabbit skeletal T-tubule membranes in terms of the affinity and the selectivity to the binding sites for the three classical calcium antagonists, such as dihydropyridines, phenylalkylamines and benzothiazepines. DTZ323, like diltiazem and clentiazem, exhibited complete and concentration-dependent inhibition of d-cis-[3H]diltiazem binding to the membrane with a slope factor close to unity. Ki values indicated that DTZ323 (Ki = 6.6 +/- 0.6 nM, mean +/- S.E., n = 4) was 48 times and 9 times more potent than diltiazem and clentiazem, respectively. DTZ323 partially inhibited the specific binding of a dihydropyridine ligand, (+)-[3H]PN200-110, at 37 degrees C. The equilibrium saturation study showed that DTZ323 reduces the affinity for the (+)-[3H]PN200-110 binding in a concentration-dependent manner with a slight decrease in the density of the binding sites. DTZ323 also inhibited the specific binding of a phenylalkylamine ligand, (-)-[3H]D888, completely as did diltiazem. DTZ323 (1 microM) had no effect on the dissociation rate of d-cis-[3H]diltiazem at 2 degrees C, whereas 30 microM verapamil increased the dissociation rate, which suggested that DTZ323 inhibits the specific binding of d-cis-[3H]diltiazem in a manner similar to other competitive ligands for the benzothiazepine binding site. These results indicate that DTZ323 is a selective ligand for the 1,5-benzothiazepine binding site with the highest affinity among the diltiazem derivatives.


Subject(s)
Calcium Channel Blockers/metabolism , Calcium Channels/metabolism , Diltiazem/analogs & derivatives , Muscle, Skeletal/metabolism , Animals , Binding Sites , Binding, Competitive , Calcium Channel Blockers/pharmacology , Calcium Channels, L-Type , Diltiazem/metabolism , Diltiazem/pharmacology , Isradipine/metabolism , Male , Rabbits , Verapamil/analogs & derivatives , Verapamil/metabolism
12.
Mol Pharmacol ; 51(2): 262-8, 1997 Feb.
Article in English | MEDLINE | ID: mdl-9203631

ABSTRACT

To determine whether 1,5-benzothiazepine Ca2+ channel blocker approaches its binding domain within the cardiac L-type Ca2+ channel from inside or outside of the membrane, we tested the effects of a novel potent 1,5-benzothiazepine derivative (DTZ323) and its quaternary ammonium derivative (DTZ417) on guinea pig ventricular myocytes by using the whole-cell patch-clamp technique. The extracellular application of DTZ417 suppressed the L-type Ca2+ channel currents (I[Ca(L)]) with an IC50 value of 1.2 +/- 0.02 microM, which was close to the IC50 value of diltiazem (0.63 +/- 0.01 microM). The suppression of I[Ca(L)] by DTZ417 was voltage and use dependent but lacked tonic block, which allowed us to investigate the onset of the effect on I[Ca(L)] by changing the holding potential (HP) from -90 to -50 mV in the presence of DTZ417. DTZ417 did not have significant effects on I[Ca(L)] at an HP of -90 mV. At -50 mV, DTZ417 (50 microM) applied from the extracellular side completely suppressed I[Ca(L)], whereas it had no effect from the intracellular side. DTZ323 (1 microM) also inhibited I[Ca(L)] only from the extracellular side, without any effects by the intracellular application of < or = 10 microM. However, a quaternary phenylalkylamine derivative, D890 (0.1 mM), acted only from the intracellular side. These results suggest that in contrast to the phenylalkylamine binding site, in cardiac myocytes the 1,5-benzothiazepine binding site is accessible from the extracellular side of the L-type Ca2+ channel.


Subject(s)
Calcium Channels/drug effects , Diltiazem/analogs & derivatives , Heart/drug effects , Thiazepines/pharmacology , Animals , Cell Membrane/drug effects , Diltiazem/pharmacology , Dose-Response Relationship, Drug , Guinea Pigs , Male
13.
Eur J Pharmacol ; 319(1): 101-7, 1997 Jan 14.
Article in English | MEDLINE | ID: mdl-9030904

ABSTRACT

To examine whether the modulation of the 1,4-dihydropyridine-binding by diltiazem derivatives, which has been shown in cardiac and skeletal muscle membranes, takes place in intact cardiac myocytes, effects of diltiazem on the specific binding of [3H](+)-PN200-110 to freshly isolated adult rat ventricular myocytes were investigated in normal Tyrode solution at 37 degrees C. Diltiazem consistently potentiated the [3H](+)-PN200-110-binding in a concentration-dependent manner, while DTZ323 (3-(acetyloxy)-5-[2-[[2- (3,4-dimethoxyphenyl)ethyl]-methylamino]ethyl]-2,3-dihydro-2(-4 methoxyphenyl)-1,5-benzothiazepin-4-(5H)-one), a potent diltiazem derivative, inhibited it in a non-competitive manner. In saturation studies, 100 microM decreased the Kd value of the 3[H](+)-PN200-110-binding (control, 0.102 +/- 0.008 vs. diltiazem, 0.074 +/- 0.004 (nM, n = 6), P < 0.05) without significant effect on Bmax (control, 65.7 +/- 6.4 vs. diltiazem, 76.7 +/- 4.4 (fmol/mg protein, n = 6). Moreover, membrane-impermeant quaternary diltiazem also potentiated the [3H](+)-PN200-110-binding in intact myocytes. These results suggest that diltiazem modulates the 1,4-dihydro-pyridine-binding even in intact cardiac myocytes, and the binding site of diltiazem is accessible from the extracellular side of the L-type Ca2+ channels.


Subject(s)
Calcium Channel Blockers/pharmacology , Calcium Channels/metabolism , Diltiazem/pharmacology , Myocardium/metabolism , Animals , Binding Sites , Binding, Competitive , Calcium Channels/drug effects , Calcium Channels, L-Type , Isradipine/metabolism , Male , Rats , Rats, Wistar
14.
J Gen Physiol ; 108(5): 435-54, 1996 Nov.
Article in English | MEDLINE | ID: mdl-8923268

ABSTRACT

Calcium-mediated cross-signaling between the dihydropyridine (DHP) receptor, ryanodine receptor, and Na(+)-Ca2+ exchanger was examined in single rat ventricular myocytes where the diffusion distance of Ca2+ was limited to < 50 nm by dialysis with high concentrations of Ca2+ buffers. Dialysis of the cell with 2 mM Ca(2+)- indicator dye, Fura-2, or 2 mM Fura-2 plus 14 mM EGTA decreased the magnitude of ICa-triggered intracellular Ca2+ transients (Cai-transients) from 500 to 20-100 nM and completely abolished contraction, even though the amount of Ca2+ released from the sarcoplasmic reticulum remained constant (approximately 140 microM). Inactivation kinetics of ICa in highly Ca(2+)-buffered cells was retarded when Ca2+ stores of the sarcoplasmic reticulum (SR) were depleted by caffeine applied 500 ms before activation of ICa, while inactivation was accelerated if caffeine-induced release coincided with the activation of ICa. Quantitative analysis of these data indicate that the rate of inactivation of ICa was linearly related to SR Ca(2+)-release and reduced by > 67% when release was absent. Thapsigargin, abolishing SR release, suppressed the effect of caffeine on the inactivation kinetics of ICa. Caffeine-triggered Ca(2+)-release, in the absence of Ca2+ entry through the Ca2+ channel (using Ba2+ as a charge carrier), caused rapid inactivation of the slowly decaying Ba2+ current. Since Ba2+ does not release Ca2+ but binds to Fura-2, it was possible to calibrate the fluorescence signals in terms of equivalent cation charge. Using this procedure, the amplification factor of ICa-induced Ca2+ release was found to be 17.6 +/- 1.1 (n = 4). The Na(+)-Ca2+ exchange current, activated by caffeine-induced Ca2+ release, was measured consistently in myocytes dialyzed with 0.2 but not with 2 mM Fura-2. Our results quantify Ca2+ signaling in cardiomyocytes and suggest the existence of a Ca2+ microdomain which includes the DHP/ ryanodine receptors complex, but excludes the Na(+)-Ca2+ exchanger. This microdomain appears to be fairly inaccessible to high concentrations of Ca2+ buffers.


Subject(s)
Calcium Channels/physiology , Carrier Proteins/physiology , Muscle Proteins/physiology , Myocardium/chemistry , Animals , Barium/metabolism , Barium/pharmacology , Biological Transport/drug effects , Biological Transport/physiology , Buffers , Caffeine/pharmacology , Calcium/metabolism , Calmodulin-Binding Proteins/physiology , Dialysis , Enzyme Inhibitors/pharmacology , Fura-2 , Heart Ventricles/chemistry , Heart Ventricles/metabolism , Ion Channel Gating/drug effects , Ion Channel Gating/physiology , Kinetics , Myocardium/metabolism , Patch-Clamp Techniques , Phosphodiesterase Inhibitors/pharmacology , Pulsatile Flow , Rats , Ryanodine Receptor Calcium Release Channel , Sodium/metabolism , Sodium-Calcium Exchanger , Thapsigargin/pharmacology
15.
Jpn J Pharmacol ; 69(4): 343-50, 1995 Dec.
Article in English | MEDLINE | ID: mdl-8786637

ABSTRACT

We studied the effects of prolonged infusion of a selective beta 1-adrenoceptor (beta 1AR) full agonist, T-0509 [(-)-(R)-1-(3,4-dihydroxyphenyl)-2-[(3,4- dimethoxyphenethyl)amino]ethanol hydrochloride], with regard to its inotropic effect in vivo and cardiac beta AR density. The results were compared with those for isoproterenol. Continuous infusion of isoproterenol at doses of 2.5-40 micrograms/kg/hr, s.c. for 6 days shifted the dose-response curves of isoproterenol (i.v.) for LVdP/dtmax to the right and increased the ED50 values up to fourfold. Isoproterenol infusion at 40 micrograms/kg/hr reduced the density of both beta 1- and beta 2ARs by 36% and 43% respectively, in left ventricular membranes. Following 6-day infusion of T-0509 at doses sufficient to induce a positive inotropic effect (5-40 micrograms/kg/hr), the ED50 value of T-0509 (i.v.) for LVdP/dtmax was also increased up to fourfold. In contrast to isoproterenol, infusion of T-0509 caused selective down-regulation of beta 1ARs by 30% without changing the number of beta 2ARs. These results indicate that long-term application of a selective beta 1AR full agonist causes desensitization to its inotropy in vivo, with subtype-selective down-regulation of beta 1ARs in cardiac ventricles.


Subject(s)
Adrenergic beta-Agonists/pharmacology , Ethanolamines/pharmacology , Heart/drug effects , Receptors, Adrenergic, beta-1/drug effects , Animals , Blood Pressure/drug effects , Dose-Response Relationship, Drug , Down-Regulation , Heart Rate/drug effects , Male , Rats , Rats, Sprague-Dawley
16.
Br J Pharmacol ; 116(1): 1635-43, 1995 Sep.
Article in English | MEDLINE | ID: mdl-8564230

ABSTRACT

1. The relative proportions of beta 1- and beta 2-adrenoceptors were determined by radioligand binding studies in three different rat myocardial preparations: membranes prepared from rat ventricle (ventricular membranes), membranes prepared from rat isolated ventricular myocytes (myocyte membranes), and myocytes isolated from rat ventricle (myocytes). 2. Competition experiments using CGP 20712A or ICI 118,551 with [125I]-iodocyanopindolol ([125I]-ICYP) revealed high- and low-affinity binding sites in ventricular membranes. The concentration at which each beta-antagonist occupied 100% of its high-affinity binding sites was 300 nM for CGP 20712A (beta 1-adrenoceptor) and 50 nM for ICI 118,551 (beta 2-adrenoceptor). 3. The density of high-affinity (beta 1-adrenoceptor) and low-affinity (beta 2-adrenoceptor) binding sites for CGP 20712A was measured by a saturation experiment using [125I]-ICYP in the presence and absence of 300 nM CGP 20712A. In ventricular membranes, the proportions of high-affinity and low-affinity binding sites for CGP 20712A were 73% and 27%, respectively, whereas in myocyte membranes, the corresponding figures were 90% and 10%, respectively. The density of low-affinity binding sites for CGP 20712A in ventricular membranes, defined as [125I]-ICYP-specific binding in the presence of 300 nM CGP 20712A, was decreased by addition of 50 nM ICI 118,551, whereas that in myocyte membranes was not affected. 4. In myocytes, specific binding of [125I]-ICYP and [3H]-CGP 12177 was not detected by saturation experiments performed in the presence of 300 nM CGP 20712A. 5 In myocytes, the activation of adenylate cyclase caused by beta2-adrenoceptors was not detected in the presence of 10 nM, 100 nM or 1000 nM CGP 20712A, which selectively antagonized beta1-adrenoceptors.Furthermore, the concentration-response curve for isoprenaline-stimulated cyclic AMP accumulation was not shifted by 10 nm or 100 nM ICI 118,551, which selectively antagonized beta2-adrenoceptors, but was shifted to the right by 1000 nM ICI 118,551.6 These results indicate that beta2-adrenoceptors are not present on rat ventricular myocytes and that beta2-adrenoceptor stimulation does not cause any detectable production of cyclic AMP. We conclude that only beta1-adrenoceptors exist on rat ventricular myocytes.


Subject(s)
Adrenergic beta-Antagonists/pharmacology , Myocardium/ultrastructure , Receptors, Adrenergic, beta-1/analysis , Receptors, Adrenergic, beta-2/analysis , Adrenergic beta-Agonists/pharmacology , Adrenergic beta-Antagonists/metabolism , Animals , Binding, Competitive , Cyclic AMP/biosynthesis , Cyclic AMP/metabolism , Heart Ventricles/cytology , Imidazoles/metabolism , Imidazoles/pharmacology , Iodocyanopindolol , Isoproterenol/pharmacology , Kinetics , Male , Membranes/metabolism , Membranes/ultrastructure , Myocardium/cytology , Myocardium/metabolism , Pindolol/analogs & derivatives , Pindolol/metabolism , Pindolol/pharmacology , Propanolamines/metabolism , Propanolamines/pharmacology , Radioligand Assay , Rats , Rats, Sprague-Dawley , Receptors, Adrenergic, beta-1/drug effects , Receptors, Adrenergic, beta-1/metabolism , Receptors, Adrenergic, beta-2/drug effects , Receptors, Adrenergic, beta-2/metabolism , Stimulation, Chemical , Tritium
17.
Eur J Pharmacol ; 264(2): 223-6, 1994 Oct 24.
Article in English | MEDLINE | ID: mdl-7851487

ABSTRACT

We report here the quantitative evaluation of binding density (Bmax) of [3H](+)-PN200-110 in aortic membranes obtained from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats. At both 4 and 13 weeks of age, there were no significant differences in Bmax and the dissociation constants (Kd) of [3H](+)-PN200-110 binding between SHR and WKY rat aortas. Irrespective of strain, the Kd increased and the Bmax decreased with age. These results suggest that the number of Ca2+ channels in aortas of SHR and WKY rats are not significantly different, even when hypertension is established in SHR.


Subject(s)
Aorta, Abdominal/metabolism , Aorta, Thoracic/metabolism , Hypertension/metabolism , Isradipine/metabolism , Muscle, Smooth, Vascular/metabolism , Aging/metabolism , Animals , Calcium Channels/metabolism , Disease Models, Animal , Male , Radioligand Assay , Rats , Rats, Inbred SHR , Rats, Inbred WKY
18.
Br J Pharmacol ; 113(2): 451-6, 1994 Oct.
Article in English | MEDLINE | ID: mdl-7834195

ABSTRACT

1. The effects of elevation of extracellular K+ concentration ([K+]o) on the negative inotropic potencies of three representative calcium antagonists, diltiazem, verapamil and nifedipine, were investigated in guinea-pig papillary muscle preparations. 2. The negative inotropic effect of diltiazem was potentiated 110 fold when [K+]o was raised from 2.7 mM to 12.7 mM. The effect of verapamil was also potentiated to a lesser extent, but that of nifedipine was not affected. 3. Resting membrane potentials in ventricular muscles were about -80 mV and -60 mV in 2.7 mM K+ and 12.7 mM K+, respectively. 4. To clarify the mechanism responsible for the differential potentiation of the negative inotropic effects, the blocking actions of the three calcium antagonists on the L-type Ca2+ channel current (ICa(L)) were compared at the holding potentials of -80 mV and -60 mV by the whole-cell patch-clamp technique. 5. The use-dependent blocking effect of diltiazem on ICa(L) was enhanced markedly by the change in the holding potential from -80 mV to -60 mV. The effect of verapamil was also enhanced to a lesser extent but that of nifedipine was not affected in this range of depolarization. 6. The differential effects of the [K+]o elevation on the negative inotropic potencies of the three calcium antagonists are explained by the differences in voltage-dependency of their use-dependent blocking effects on ICa(L). 7. The properties of diltiazem and verapamil observed in this study may contribute to their protective effects on the ischaemic myocardium, without affecting the normal myocardium.


Subject(s)
Calcium Channel Blockers/pharmacology , Calcium Channels/metabolism , Myocardial Contraction/drug effects , Myocardium/metabolism , Animals , Calcium Channels/drug effects , Cell Polarity/drug effects , Diltiazem/pharmacology , Guinea Pigs , In Vitro Techniques , Male , Membrane Potentials/drug effects , Myocardium/cytology , Nifedipine/pharmacology , Papillary Muscles/cytology , Papillary Muscles/drug effects , Papillary Muscles/metabolism , Potassium/pharmacology , Verapamil/pharmacology
19.
Biol Pharm Bull ; 17(8): 1023-7, 1994 Aug.
Article in English | MEDLINE | ID: mdl-7820101

ABSTRACT

The affinities for beta-adrenoceptors, the subtype-selectivity and the agonistic effectiveness of T-0509 (a catechol derivative of denopamine) and colterol (N-tert-butylnoradrenaline; Col) were compared with those of other beta-agonists using a binding assay method. Specific binding of [3H]dihydroalprenolol (3H-DHA) to guinea pig left ventricular and lung membranes was saturable, and Scatchard and Hill analyses suggested that 3H-DHA bound to both membranes with a single population of binding sites with no binding site cooperativity. Addition of 5'-guanylylimidodiphosphate (GppNHp, 30 microM) led to a rightward shift of the 3H-DHA binding displacement curves of T-0509 and Col in both membranes, and the degree of shift was similar to that of full agonists such as isoproterenol (Iso), adrenaline (Adr) and noradrenaline (NA). Both T-0509 and Col were thus considered to be full agonists at both beta 1- and beta 2-adrenoceptors, respectively, unlike denopamine and procaterol. T-0509 and Col showed considerably high affinity for both beta 1- and beta 2-adrenoceptors, and T-0509, like denopamine, was as selective for the beta 1-subtype as NA (4.5-fold compared with Iso as a non-selective agonist), whereas Col was more selective for the beta 2-subtype than Adr (4.5-fold compared with Iso).


Subject(s)
Adrenergic beta-Agonists/pharmacology , Catechols/metabolism , Ethanolamines/pharmacology , Nordefrin/analogs & derivatives , Receptors, Adrenergic, beta-1/metabolism , Receptors, Adrenergic, beta-2/metabolism , Animals , Dihydroalprenolol , Guanine Nucleotides/pharmacology , Guanylyl Imidodiphosphate/pharmacology , Guinea Pigs , Kinetics , Lung/metabolism , Male , Myocardium/metabolism , Nordefrin/pharmacology , Propranolol/pharmacology
20.
Biol Pharm Bull ; 17(7): 907-10, 1994 Jul.
Article in English | MEDLINE | ID: mdl-8000374

ABSTRACT

Three types of calcium antagonists, diltiazem, verapamil and nicardipine, were separately infused into Sprague-Dawley (SD) rats (under pentobarbital anesthesia n = 5) through the left femoral vein at four different flow rates. Mean arterial blood pressure, heart rate and the concentration of plasma catecholamines (CAs), epinephrine (E), norepinephrine (NE) and dopamine (DA), were measured for each calcium antagonist, and the correlations between them were studied. Blood samples were collected within the infusion from common jugular vein. Plasma concentrations of CAs were determined by a HPLC-ethylenediamine condensation reaction-peroxyoxalate chemiluminescence detection system (HPLC-ED-PO-CL). The plasma concentration of CAs increased corresponding to the blood pressure reduction. The reduction induced by each calcium antagonist correlated with the logarithm of plasma NE concentration. The relation was expressed as Y = -alpha log X+m (Y, blood pressure; X, concentration of plasma NE; alpha, slope; and m, intercept). The correlation coefficients (rs) were -0.950 (diltiazem), -0.975 (verapamil) and -0.978 (nicardipine) (versus -0.734 for control). The alpha for nicardipine (108.4) was greater than those of diltiazem (85.4) and verapamil (80.8) (versus 31.0 for control), meaning that blood pressure reduction was greater in the case of nicardipine than diltiazem and verapamil, with an identical increment of plasma NE concentration. These data indicate that the contribution of the sympathetic nervous system to maintaining blood pressure reduced by nicardipine is less than that observed following the infusion of diltiazem and verapamil. Similar good inverse correlations between blood pressure and the logarithm of plasma concentration of E were observed with the three drugs infused (r = -0.928, -0.966, and -0.948 for diltiazem, verapmil and nicardipine, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Blood Pressure/drug effects , Calcium Channel Blockers/pharmacology , Catecholamines/blood , Animals , Diltiazem/pharmacology , Heart Rate/drug effects , Nicardipine/pharmacology , Rats , Rats, Sprague-Dawley , Verapamil/pharmacology
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