Taiji Furukawa

Voltage and pH dependent block of cloned N-type Ca2+ channels by amlodipine

Two types of Ca2+ channel α1‐subunits were co‐expressed in Xenopus oocytes with the Ca2+ channel α2‐ and β1‐subunits. The Ba2+ current through the α1Cα2β and the α1Bα2β channels had electrophysiological and pharmacological properties of L‐ and N‐type Ca2+ channels, respectively. Amlodipine had a strong blocking action on both the L‐type and N‐type Ca2+ channels expressed in the oocyte. The potency of the amlodipine block on the N‐type Ca2+ channel was comparable to that on the L‐type Ca2+ channel. At −100mV holding potential, the IC50 values for amlodipine block on the L‐type and N‐type Ca2+ channel were 2.4 and 5.8μm, respectively. The blocking action of amlodipine on the N‐type Ca2+ channel was dependent on holding potential and extracellular pH, as has been observed with amlodipine block on the L‐type Ca2+ channel. A depolarized holding potential and high pH enhanced the blocking action of amlodipine. The time course of block development by amlodipine was similar for L‐type and N‐type Ca2+ channels. However, it was slower than the time course of block development by nifedipine for the L‐type Ca2+ channel.

DIFFERENTIAL INTERACTIONS OF THE C TERMINUS AND THE CYTOPLASMIC I-II LOOP OF NEURONAL CA2+ CHANNELS WITH G-PROTEIN ALPHA AND BETA GAMMA SUBUNITS. II . EVIDENCE FOR DIRECT BINDING

The present study was designed to obtain evidence for direct interactions of G-protein α (Gα) and βγ subunits (Gβγ) with N- (α1B) and P/Q-type (α1A) Ca2+ channels, using synthetic peptides and fusion proteins derived from loop 1 (cytoplasmic loop between repeat I and II) and the C terminus of these channels. For N-type, prepulse facilitation as mediated by Gβγ was impaired when a synthetic loop 1 peptide was applied intracellularly. Receptor agonist-induced inhibition of N-type as mediated by Gα was also impaired by the loop 1 peptide but only when applied in combination with a C-terminal peptide. For P/Q-type channels, by contrast, the Gα-mediated inhibition was diminished by application of a C-terminal peptide alone. Moreover, in vitro binding analysis for N- and P/Q-type channels revealed direct interaction of Gα with C-terminal fusion proteins as well as direct interaction of Gβγ with loop 1 fusion proteins. These findings define loop 1 of N- and P/Q-type Ca2+ channels as an interaction site for Gβγ and the C termini for Gα.

Comprehensive analysis of the renin-angiotensin gene polymorphisms with relation to hypertension in the Japanese

Background Components of the renin–angiotensin (R–A) system have been repeatedly investigated as candidate genes for essential hypertension. In particular, suggestive or significant association has been detected in some studies for the angiotensinogen M235T, angiotensin I-converting enzyme I/D, angiotensin II type 1 receptor A1166C, and aldosterone synthase C-344T polymorphisms, although the results remain inconclusive. Objective and Methods To evaluate the importance of these candidate genes for hypertension, we undertook an extensive association study in the Japanese. This case–control study was conducted in a total of 1476 individuals using the four R–A gene polymorphisms. In the assessment of genotyping data, 843 hypertensive subjects were divided into three case subgroups according to severity of hypertension, while 633 normotensive subjects divided into two control subgroups by the age of enrollment. Each subgroup was further divided by sex. Subsequently, the presence of synergy (or gene–gene interaction) was evaluated among four R–A gene polymorphisms. Results No significant association was observed between the individual R–A gene polymorphisms and hypertension status in our case–control study. The results were almost unchanged when severity of hypertension, sex-specificity, and synergy were taken into account. Conclusions Despite a relatively large number of subjects, we did not find significant evidence for disease association in the Japanese population. Given confounding factors in the case–control strategy, the lack of association does not exclude the relevance of the R–A genes to hypertension. Further investigation needs to be performed in large-scale populations, where the use of not only hypertension status, but also ‘intermediate’ phenotypes would be useful.

Angiotensin-converting enzyme inhibition improves defective angiogenesis in the ischemic limb of spontaneously hypertensive rats.

OBJECTIVES Natural angiogenesis has been shown to be impaired in spontaneously hypertensive rats (SHR). The purpose of this study was to determine whether pathological angiogenesis in the setting of tissue ischemia is also impaired in SHR, and to what extent it is modified by angiotensin-converting enzyme (ACE) inhibition. METHODS Ischemia was induced in the hindlimb of SHR by excision of the femoral artery, after which the animals were randomly assigned to receive low-dose perindopril (sub-antihypertensive, 0.2 mg/kg/day), high-dose perindopril (antihypertensive, 2.0 mg/kg/day), or vehicle for 3 weeks. Wistar-Kyoto rats (WKY) with femoral artery excision served as a control group. RESULTS Tissue ACE activity in SHR was significantly increased compared to WKY (49.4+/-6.2 vs. 34.0+/-14.2 IU/mg, P<0.01). Administration of perindopril significantly reduced ACE activity in SHR (low dose: 12.4+/-2.3; high dose: 11.0+/-2.1 IU/mg, P<0.005). Angiogenesis of the ischemic limb muscles was significantly impaired at 4 weeks in SHR versus WKY as indicated by the lower capillary density in the former (364.5+/-43.0 vs. 463.8+/-63.0/mm(2), P<0.05) as well as the reduced hindlimb perfusion assessed by laser Doppler imaging (0.86+/-0.08 vs. 1.03+/-0.09, P<0.05). Administration of perindopril significantly augmented both the capillary density (low dose: 494.3+/-69.8; high dose: 543.9+/-76.9/mm(2), P<0.005) and the limb perfusion (low dose: 1.06+/-0.15; high dose: 1.05+/-0.12, P<0.05) of the ischemic limb in SHR. CONCLUSIONS These findings indicate that pathological angiogenesis in the setting of tissue ischemia is impaired in SHR compared with WKY, and that this impairment can be reversed by ACE inhibition. The angiogenic properties of an ACE inhibitor may benefit patients with essential hypertension presenting with lower limb vascular insufficiency.

Differential blocking action of dihydropyridine Ca2+ antagonists on a T-type Ca2+ channel (alpha1G) expressed in Xenopus oocytes.

Recent reports show that efonidipine, a dihydropyridine Ca2+ antagonist, has blocking action on T-type Ca2+ channels, which may produce favorable actions on cardiovascular systems. However, the effects of other dihydropyridine Ca2+ antagonists on T-type Ca2+ channels have not been investigated yet. Therefore, in this study, we examined the effects of dihydropyridine compounds clinically used for treatment of hypertension on a T-type Ca2+ channel subtype, α1G, expressed in Xenopus oocytes. These effects were compared with those on T-type Ca2+ channel. Rabbit L-type (α1Cα2/δβ1a) or rat T-type (α1G) Ca2+ channel was expressed in Xenopus oocytes by injection of cRNA for each subunit. The Ba2+ currents through expressed channels were measured by conventional 2-microelectrode voltage-clamp methods. Twelve DHPs (amlodipine, barnidipine, benidipine, cilnidipine, efonidipine, felodipine, manidipine, nicardipine, nifedipine, nilvadipine, nimodipine, nitrendipine) and mibefradil were tested. Cilnidipine, felodipine, nifedipine, nilvadipine, minodipine, and nitrendipine had little effect on the T-type channel. The blocks by drugs at 10 μM were less than 10% at a holding potential of −100 mV. The remaining 6 drugs had blocking action on the T-type channel comparable to that on the L-type channel. The blocking actions were also comparable to that by mibefradil. These results show that many dihydropyridine Ca2+ antagonists have blocking action on the α1G channel subtype. The action of dihydropyridine Ca2+ antagonists in clinical treatment should be evaluated on the basis of subtype selectivity.

Identification of R(−)‐isomer of efonidipine as a selective blocker of T‐type Ca2+ channels

Efonidipine, a derivative of dihydropyridine Ca2+ antagonist, is known to block both L‐ and T‐type Ca2+ channels. It remains to be clarified, however, whether efonidipine affects other voltage‐dependent Ca2+ channel subtypes such as N‐, P/Q‐ and R‐types, and whether the optical isomers of efonidipine have different selectivities in blocking these Ca2+ channels, including L‐ and T‐types. To address these issues, the effects of efonidipine and its R(−)‐ and S(+)‐isomers on these Ca2+ channel subtypes were examined electrophysiologically in the expression systems using Xenopus oocytes and baby hamster kidney cells (BHK tk‐ts13). Efonidipine, a mixture of R(−)‐ and S(+)‐isomers, exerted blocking actions on L‐ and T‐types, but no effects on N‐, P/Q‐ and R‐type Ca2+ channels. The selective blocking actions on L‐ and T‐type channels were reproduced by the S(+)‐efonidipine isomer. By contrast, the R(−)‐efonidipine isomer preferentially blocked T‐type channels. The blocking actions of efonidipine and its enantiomers were dependent on holding potentials. These findings indicate that the R(−)‐isomer of efonidipine is a specific blocker of the T‐type Ca2+ channel.

Differential interactions of the C terminus and the cytoplasmic I-II loop of neuronal Ca2+ channels with G-protein alpha and beta gamma subunits. I. Molecular determination.

Interactions of G-protein α (Gα) and βγ subunits (Gβγ) with N- (α1B) and P/Q-type (α1A) Ca2+ channels were investigated using the Xenopus oocyte expression system. Gi3α was found to inhibit both N- and P/Q-type channels by receptor agonists, whereas Gβ1γ2 was responsible for prepulse facilitation of N-type channels. L-type channels (α1C) were not regulated by Gα or Gβγ. For N-type, prepulse facilitation mediated via Gβγ was impaired when the cytoplasmic I-II loop (loop 1) was deleted or replaced with the α1C loop 1. Gα-mediated inhibitions were also impaired by substitution of the α1C loop 1, but only when the C terminus was deleted. For P/Q-type, by contrast, deletion of the C terminus alone diminished Gα-mediated inhibition. Moreover, a chimera of L-type with the α1B loop 1 gained Gβγ-dependent facilitation, whereas an L-type chimera with the N- or P/Q-type C terminus gained Gα-mediated inhibition. These findings provide evidence that loop 1 of N-type channels is a regulatory site for Gβγ and the C termini of P/Q- and N-types for Gα.

Ethanol inhibition of Ca2+ and Na+ currents in the guinea-pig heart

The effects of ethanol on L-type Ca2+ and fast Na+ currents (ICa and INa, respectively) were examined using the whole-cell patch-clamp experiments on guinea-pig ventricular cells. At a clinically relevant concentration of 24 mM, ethanol slightly but significantly shortened the action potential duration, and reduced the ICa by 7 +/- 4% (mean +/- S.D.). This concentration of ethanol did not affect INa, but a lethal concentration of ethanol (80 mM) significantly inhibited INa by 13 +/- 5%. The voltage dependence of INa activation was not affected by ethanol, whereas the inhibitions of ICa by 80 mM ethanol and INa by 240 mM were both accompanied by a several mV shift in the channel availability curve toward more negative potentials, suggesting that the channels in the inactivated state are more susceptible to ethanol. The ICa inhibition by ethanol at clinically relevant concentrations could contribute to a negative inotropic effect, action potential shortening and development of arrhythmias, while the pathophysiological significance of ethanol inhibition of INa seems less important.

Five different profiles of dihydropyridines in blocking T-type Ca2+ channel subtypes (Cav3.1 (α1G), Cav3.2 (α1H), and Cav3.3 (α1I)) expressed in Xenopus oocytes☆

1,4-dihydropyridine (DHP) Ca(2+) antagonists have recently been shown to block T-type Ca(2+) channels, which may render favorable actions on cardiovascular systems. However, this evaluation remains to be done systematically for each T-type Ca(2+) channel subtype except for the Ca(v)3.1 (alpha(1G)) subtype. To address this issue at the molecular level, blocking effects of 14 kinds of DHPs (amlodipine, aranidipine, azelnidipine, barnidipine, benidipine, cilnidipine, efonidipine, felodipine, manidipine, nicardipine, nifedipine, nilvadipine, nimodipine, nitrendipine), which are clinically used for treatments of hypertension, on 3 subtypes of T-type Ca(2+) channels [Ca(v)3.2 (alpha(1H)), Ca(v)3.3 (alpha(1I)), and Ca(v)3.1 (alpha(1G))] were investigated in the Xenopus oocyte expression system using the two-microelectrode voltage-clamp technique. These 3 kinds (alpha(1H), alpha(1I) and alpha(1G)) of T-type channels were blocked by amlodipine, manidipine and nicardipine. On the other hand, azelnidipine, barnidipine, benidipine and efonidipine significantly blocked alpha(1H) and alpha(1G), but not alpha(1I) channels, while nilvadipine and nimodipine apparently blocked alpha(1H) and alpha(1I), but not alpha(1G) channels. Moreover, aranidipine blocked only alpha(1H) channels. By contrast, cilnidipine, felodipine, nifedipine and nitrendipine had little effects on these subtypes of T-type channels. The result indicates that the blockade of T-type Ca(2+) channels by derivatives of DHP Ca(2+) antagonist was selective for the channel subtype. Therefore, these selectivities of DHPs in blocking T-type Ca(2+) channel subtypes would provide useful pharmacological and clinical information on the mode of action of the drugs including side-effects and adverse effects.

First Report of Metallo-β-Lactamase NDM-5-Producing Escherichia coli in Japan

Reports of carbapenemase-producing carbapenem-resistant Enterobacteriaceae are increasing worldwide ([1][1]). Among the newly emerged carbapenemases, New Delhi metallo-β-lactamase 1 (NDM-1) represents the latest threat to public health. Since it was first described in 2009 ([2][2]), NDM-producing