Hisanori Hazama

17β-Estradiol inhibits the voltage-dependent L-type Ca2+ currents in aortic smooth muscle cells

To elucidate the mechanisms of estrogens-induced relaxation effects on vascular smooth muscle cells, the effects of estrogens and the related hormones were examined in cultured rat thoracic aortic smooth muscle cell lines (A7r5), using the whole-cell voltage clamp technique. The patch pipette was filled with 140 mM CsCl- or KCl-containing internal solution. With CsCl-internal solution, 17β-estradiol and synthetic estrogens, ethynylestradiol and diethylstilbestrol (0.1–30 μM) inhibited the Ba2+ inward current (IBa) through the voltage-dependent L-type Ca2+ channel in a concentration-dependent and reversible manner. The potency of the inhibitory effects on IBa was 17β-estradiol < ethynylestradiol < diethylstilbestrol. 17β-Estradiol (10 μM) appeared to reduce the maximal conductance of IBa with only a slight shift of voltage-dependency of inactivation and to affect IBa in a use-independent fashion. On the other hand, testosterone and progesterone (30 μM) failed to affect IBa. At a holding potential of −40 mV, both vasopressin and endothelin-1 (100 nM) activated a long-lasting inward current. After endothelin-1 (100 nM) activated the current, the additional application of vasopression (100 nM) could not induce it furthermore, suggesting that each agonist activates the same population of the channels. The reversal potential of the current was about 0 mV and was not significantly altered by replacement of [Cl−]i or [Cl−]0 and the inward current was also observed even when extracellular cations are Ca2+, proposing that it was a Ca2+-permeable non-selective cation channel (IN.S.). La3+ or Cd2+ (1 mM) completely abolished IN.S., however, nifedipine (10 μM) failed to inhibit it at all. Diethylstilbestrol (1–30 μM) suppressed the IN.S. evoked by both endothelin-1 and vasopressin in a concentration-dependent manner, while 17β-estradiol, ethynylestradiol, progesterone and testosterone (30 μM) failed to inhibit it significantly. In addition, at a holding potential of +0 mV, 17β-estradiol by itself did not affect the holding currents, and did not inhibit K+ currents evoked by endothelin-1 or vasopressin, possibly due to the Ca2+ release from the storage sites. These results suggest that 17β-estradiol may play a role in regulating vascular tone, selectively by inhibiting the voltage-dependent L-type Ca2+ current in vascular smooth muscle cells.

Antiarrhythmic effect and its underlying ionic mechanism of 17β-estradiol in cardiac myocytes

The effects of oestrogens on action potential and membrane currents were examined in single guinea‐pig atrial myocytes. 17β‐estradiol (3–10 μM) shortened the action potential duration without significant changes in the resting membrane potential. E‐4031 (1 μM) markedly prolonged the action potential duration and induced early afterdepolarization, and 17β‐estradiol (10 μM) abolished it. When cells were perfused in isoproterenol‐containing solution, action potentials due to abnormal automaticity caused by membrane depolarization developed, and were also inhibited by 17β‐estradiol. Under voltage clamp conditions, the voltage‐dependent Ca2+ currents consisted of both T‐(ICa.T) and L‐type (ICa.L). 17β‐estradiol reduced ICa.L concentration‐dependently, while it (10 μM) suppressed ICa.T only by approximately 10%. 17β‐estradiol did not affect time courses of ICa.L inactivation, but it shifted the steady‐state inactivation curve to more negative potentials. 17β‐estradiol (10 μM) did not affect the time‐dependent K+ current (IK), referred to as IKr and IKs, and inwardly rectifying K+ current. However, 17β‐estradiol (30 μM) or diethylstilbestrol (10 μM) inhibited K+ currents. DES and ethinylestradiol (EES) also suppressed ICa.L, but testosterone and progesterone failed to inhibit ICa.L. The potency of the inhibitory effect on ICa.L was DES>EES>17β‐estradiol. 17β‐estradiol and DES also inhibited the cyclic AMP‐enhanced ICa.L, but cyclic GMP in the pipette or pretreatment of L‐NAME could not block the effects of oestrogen on ICa.L. These results suggest that oestrogen specifically has antiarrhythmic effects, possibly by acting the L‐type Ca2+ channels. The antiarrhythmic effects of oestrogens may contribute to the cardioprotective actions of oestrogens.

Regional Differences in Transient Outward Current Density and Inhomogeneities of Repolarization in Rabbit Right Atrium

BACKGROUND Recent experimental and clinical studies on atrial flutter have demonstrated that the crista terminalis (CT) plays an important role in the genesis of atrial reentry. To elucidate the underlying mechanism of its role, we characterized the electrophysiological repolarization properties of CT cells by comparing them with those of the pectinate muscles (PM). METHODS AND RESULTS After action potential properties of both regions were compared by conventional microelectrode technique in multicellular atrial tissues, the whole-cell clamp experiments were applied in atrial cells isolated from both regions. Action potential duration (APD) was more prolonged in CT than in PM in multicellular preparations (APD90 77 +/- 5 ms versus 52 +/- 8 ms at 1 Hz, P < .01), though the other properties did not differ significantly. Similarly, in isolated atrial cells, APD was more prolonged in CT cells than in PM cells (APD90 63 +/- 7 ms versus 41 +/- 6 ms at 0.1 Hz, P < .01). Isolated single cells were larger in CT than in PM. The whole-cell clamp recordings showed no definite distinctions in the density of the voltage-dependent L-type Ca2+ current and the inwardly rectifying K+ current between these cells but revealed a significant reduction of the density of the 4-aminopyridine-sensitive transient outward current (Ito) in CT cells compared with that in PM cells (6.3 +/- 0.7 pA/pF versus 10.3 +/- 0.8 pA/pF at +20 mV, P < .05). However, no differences in the kinetics or the voltage dependence of Ito were observed between the cells. The time course of recovery from inactivation of Ito was also similar in both types of cells. CONCLUSIONS These results suggest that the preferential reduction in the density of Ito in the CT cells could contribute to prolong their APD, which may be related to the genesis of atrial reentry.

Effects of Extracellular pH on Receptor‐Mediated Ca2+ Influx in A7r5 Rat Smooth Muscle Cells: Involvement of Two Different Types of Channel

1 The effects of extracellular pH (pHo) on receptor (vasopressin or endothelin‐l)‐mediated Ca2+ entry and Ca2+‐permeable channels were investigated in aortic smooth muscle cells (A7r5) from rat embryonic thoracic aorta. Intracellular Ca2+ ([Ca2+]i) was measured using fura‐2 AM and whole‐cell voltage clamp techniques were employed. 2 Vasopressin and endothelin‐1 (100 nM) in the presence of nicardipine (10 μM) evoked a sustained rise in [Ca2+]i due to calcium entry. Extracellular acidosis decreased receptor (vasopressin or endothelin‐l)‐mediated Ca2+ entry, while extracellular alkalosis potentiated it. 3 Depletion of intracellular Ca2+ stores with thapsigargin (1 μM) also evoked Ca2+ entry activated by emptying of intracellular Ca2+ stores (capacitative Ca2+ entry). Extracellular acidosis decreased this capacitative Ca2+ entry, while extracellular alkalosis potentiated it. 4 Under voltage‐clamp conditions with Cs+ internal solution, vasopressin and endothelin‐1 activated non‐selective cation currents (Icat). Ba2+ or Ca2+ were also charge carriers of Icat. Reducing the pHo inhibited Icat, while increasing pHo potentiated it in a reversible manner. 5 Intracellular pH (pHi) changes did not cause the same marked effects as pHo changes, and a high concentration of Hepes (50 mM) in the patch pipette did not inhibit the effects of pHo on Icat. 6 Similar results were obtained when Icat was activated by GTPγS (1 mM) applied through the patch pipette, even in the absence of agonists, probably because of direct activation of GTP‐binding proteins coupled to the receptors. 7 In cells treated with thapsigargin, addition of Ca2+ to the bath solution induced Ca2+‐dependent K+ currents activated by capacitative Ca2+ entry. However, no measurable ionic currents activated by capacitative Ca2+ entry (ICRAC) were observed under conditions with Cs+ internal solution and EGTA (5 mM), although vasopressin still activated Icat. 8 These results suggest that the contractile agonists vasopressin and endothelin‐1 evoke Ca2+ entry through two different types of Ca2+‐permeable channel (Icat and ICRAC) and pHo affects these channels, which may modulate receptor‐mediated Ca2+ influx in A7r5 cells. Thus, pH‐induced changes of these channels may play a pathophysiological role in the control of receptor‐mediated contractions.

Troglitazone Inhibits Voltage-Dependent Calcium Currents in Guinea Pig Cardiac Myocytes

BACKGROUND It has been suggested that intracellular Ca2+ overload in cardiac myocytes leads to the development of diabetic cardiomyopathy. Troglitazone, an insulin-sensitizing agent, is a promising therapeutic agent for diabetes and has been shown to prevent diabetes-induced myocardial changes. To elucidate the underlying mechanism of troglitazone action on cardiac myocytes, the effects of troglitazone on voltage-dependent Ca2+ currents were examined and compared with classic Ca2+ antagonists (verapamil and nifedipine). METHODS AND RESULTS Whole-cell voltage-clamp techniques were applied in single guinea pig atrial myocytes. Under control conditions with CsCl internal solution, the voltage-dependent Ca2+ currents consisted of both T-type (ICa,T) and L-type (ICa,L) Ca2+ currents. Troglitazone effectively reduced the amplitude of ICa,L in a concentration-dependent manner. Troglitazone also suppressed ICa,T, but the effect of troglitazone on ICa,T was less potent than that on ICa,L. The current-voltage relationships for ICa,L and the reversal potential for ICa,L were not altered by troglitazone. The half-maximal inhibitory concentration of troglitazone on ICa,L measured at a holding potential of -40 mV was 6.3 micromol/L, and 30 micromol/L troglitazone almost completely inhibited ICa,L. Troglitazone 10 micromol/L did not affect the time courses for inactivation of ICa,L and inhibited ICa,L mainly in a use-independent fashion, without shifting the voltage-dependency of inactivation. This effect was different from those of verapamil and nifedipine. Troglitazone also reduced isoproterenol- or cAMP-enhanced ICa,L. CONCLUSIONS These results demonstrate that troglitazone inhibits voltage-dependent Ca2+ currents (T-type and L-type) and then antagonizes the effects of isoproterenol in cardiac myocytes, thus possibly playing a role in preventing diabetes-induced intracellular Ca2+ overload and subsequent myocardial changes.

Inhibitory effects of ω‐3 polyunsaturated fatty acids on receptor‐mediated non‐selective cation currents in rat A7r5 vascular smooth muscle cells

The effects of ω‐3 polyunsaturated fatty acids on receptor‐mediated non‐selective cation current (Icat) and K+ current were investigated in aortic smooth muscle cells from foetal rat aorta (A7r5 cells). The whole‐cell voltage clamp technique was employed. With a K+‐containing solution, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA, 30 μm) produced an outward current at a holding potential of −40 mV. This response was inhibited by tetraethylammonium (20 mm) or Cs+ in the patch pipette solution, and the reversal potential of the EPA‐induced current followed the K+ equilibrium potential in a near Nernstian manner. Under conditions with a Cs+‐containing pipette solution, both vasopressin and endothelin‐1 (100 nm) induced a long‐lasting inward current at a holding potential of −60 mV. The reversal potential of these agonist‐induced currents was about +0 mV, and was not significantly altered by the replacement of the extracellular or intracellular Cl− concentration, suggesting that the induced current was a cation‐selective current (Icat). La3+ and Cd2+ (1 mm) completely abolished these agonist‐induced Icat, but nifedipine (10 μm) failed to inhibit it significantly. ω‐3 polyunsaturated fatty acids (3100 μm), EPA, DHA and docosapentaenoic acids (DPA), inhibited the agonist‐induced Icat in a concentration‐dependent manner. The potency of the inhibitory effect was EPA>DHA>DPA, and the half maximal inhibitory concentration (IC50) of EPA was about 7 μm. Arachidonic and linoleic acids (10, 30 μm) showed a smaller inhibitory effect compared to ω‐3 fatty acids. Also, oleic and stearic acids (30 μm) did not show a significant inhibitory effect on Icat. A similar inhibitory action of EPA was observed when Icat was activated by intracellularly applied GTPγS in the absence of agonists, suggesting that the site of action of ω‐3 fatty acids is not located on the receptor. These results demonstrate that ω‐3 polyunsaturated fatty acids can activate a K+ current and also effectively inhibit receptor‐mediated non‐selective cation currents in rat A7r5 vascular smooth muscle cells. Thus, the data suggest that ω‐3 fatty acids may play an important role in the regulation of vascular tone.

Extracellular Mg2+ inhibits receptor-mediated Ca2+-permeable non-selective cation currents in aortic smooth muscle cells

The effects of extracellular Mg2+ on receptor-mediated Ca(2+)-permeable non-selective cation currents were investigated in a cultured aortic smooth muscle cell line (A7r5) from rat thoracic aorta, using the whole-cell voltage-clamp technique. Under the Cs(+)-containing internal solution, both vasopressin and endothelin-1 (100 nM) activated a long-lasting inward current with a high noise level. The reversal potential of these agonists-induced current was approximately +0 mV, and was not significantly altered by the replacement of [Cl-]i or [Cl-]o, suggesting that the inward current was a cation-selective channel. La3+ and Cd2+ (1 mM) almost completely abolished the vasopressin or endothelin-induced non-selective cation current; however, nifedipine (10 microM) failed to inhibit it significantly. Extracellular Mg2+ (3-20 mM) also markedly inhibited the vasopressin- or endothelin-induced non-selective cation current in a concentration-dependent manner. When a non-hydrolysable GTP-analogue, GTP gamma S (1 mM), was applied from the patch pipette, the non-selective cation current was gradually activated even in the absence of agonist (vasopressin or endothelin-1), probably due to the direct activation of GTP-binding proteins coupled to the receptors. Extracellular Mg2+ (3-20 mM) also suppressed the activation of non-selective cation current induced by GTP gamma S, suggesting that the inhibitory sites of Mg2+ are not located on the receptors. These results suggest that extracellular Mg2+ inhibits receptor-mediated non-selective cation current, which may contribute to the relaxation effects of Mg2+ in vascular smooth muscle cells.

Influence of cellular incorporation of n-3 eicosapentaenoic acid on intracellular Ca2+ concentration and membrane potential in vascular smooth muscle cells

Long-term treatment with n-3 eicosapentaenoic acid (EPA) has been shown to exert hypotensive effects and have beneficial effects on atherosclerosis. To elucidate one of the underlying mechanisms of these effects, intracellular calcium concentration [Ca2+]i, and resting membrane potential were measured in rat vascular smooth muscle cells (A7r5 cell) treated with EPA, using Ca2+-sensitive dye fura-2 AM and the patch clamp technique. The alterations in fatty acid compositions of phospholipids and cell migration after treatment with EPA (30 microM) for 6 h-7 days were also examined. After treating cells with EPA, the EPA and DPA (docosapentaenoic acid) content of the phospholipid fraction (mol.%) increased in a time-dependent manner. Alternatively, arachidonic acid (AA) decreased, and then the ratio of EPA and AA (EPA/AA) increased significantly. The resting [Ca2+]i decreased from 170 +/- 46 nM (n = 16) in control cells to 123 +/- 29 nM (n = 16) in cells treated with EPA (30 microM) for 7 days. Vasopressin (100 nM), endothelin-1 (100 nM) and platelet-derived growth factor (PDGF 5 ng/ml) evoked an initial peak of [Ca2+]i, followed by a smaller sustained rise of [Ca2+]i in the presence of extracellular Ca2+. In EPA-treated cells, both the peak and the sustained rise of [Ca2+]i induced by these agonists decreased in comparison to the control cells. EPA treatment also decreased the transient [Ca2+]i rise evoked by these agonists in the absence of extracellular Ca2+. Under the current clamp condition, resting membrane potential was significantly higher in EPA-treated cells (-49.8 +/- 10.4 mV, n = 41) than in control cells (-44.6 +/- 7.4 mV, n = 41, P < 0.05), and the input resistance of the cell was lower in EPA-treated cells, while cell size and capacitance were not statistically different. In addition, long-term treatment with EPA for 7 days significantly inhibited PDGF-induced cell migration. These results suggest that cellular incorporation of n-3 eicosapentaenoic acid attenuates intracellular mechanisms related to changes of [Ca2+]i and affects membrane potential, thereby inhibiting migration of vascular smooth muscle cells. These actions of EPA may contribute to its vasorelaxant and antiatherosclerotic effects.

Ouabainlike Compound in Hypertension Associated With Ectopic Corticotropin Syndrome

Molecular mechanisms related to sodium retention have been implicated in the pathogenesis of hypertension. It is unclear how sodium retention leads to a rise in blood pressure, but ouabainlike compound may act as a final common pathway in sodium-induced hypertension. In ectopic corticotropin syndrome, hypertension has been attributed to cortisol inactivation overload, giving rise to mineralocorticoid-type hypertension. We sequentially measured plasma and urinary levels of ouabainlike compound over 2 months to evaluate its role in the hypertensive mechanisms in a 64-year-old man with this syndrome caused by lung cancer. His data included hypokalemia and increased cortisol concentrations, corticotropin levels, and urinary 17-hydroxycorticosteroid excretion. Plasma renin activity was suppressed. Plasma and urinary levels of ouabainlike compound were markedly increased concomitantly with high blood pressure. The maximum plasma level was 40-fold the normal range of the subject. After chemotherapy, ouabainlike compound levels gradually decreased in parallel with the decline in blood pressure and rise in potassium concentration. A correlation was observed between plasma and urinary levels of ouabainlike compound (P < .05). Plasma and urinary levels of ouabainlike compound correlated with systolic (P < .01) and diastolic (P < .05) pressures, respectively. The peak of ouabainlike compound in plasma and urine coincided with that of authentic ouabain on high-performance liquid chromatography. Ouabainlike compound derived from urine inhibited [3H]ouabain binding to human erythrocytes. These findings suggest that ouabainlike compound with biological activity could partly account for hypertension in ectopic corticotropin syndrome.

Neurokinin A and Ca2+ current induce Ca(2+)-activated Cl(-) currents in guinea-pig tracheal myocytes.

1. Membrane currents were recorded by a patch clamp technique in guinea‐pig tracheal myocytes, using the whole cell mode with Cs(+) internal solution. 2. Both neurokinin A (NKA, 1 mu M) and caffeine (10 mM) evoked Ca(2+)‐activated Cl‐ currents (I[Cl(Ca)]) transiently. In Ca(2+)‐free bathing solution, the first application of NKA or caffeine elicited I[Cl(Ca)] but the second application of these substances failed to activate it. In addition, pretreatment with ryanodine in the presence of caffeine abolished the response to both NKA and caffeine whilst heparin (200 mu g ml(‐1)) only blocked the NKA‐induced response. I[Cl(Ca)] was also elicited by inositol 1,4,5‐trisphosphate (IP(3)). 3. Command voltage pulses positive to 0 mV from a holding potential of ‐60 mV activated the voltage‐dependent L‐type Ca2+ current (I(Ca,L)) and late outward current. Upon repolarization to the holding potential, slowly decaying inward tail currents were recorded. The outward current during the depolarizing pulses and the inward tail current were enhanced by Bay K 8644, but completely blocked by Cd2+ or nifedipine. Replacement of external Ca2+ with Ba2+, removal of Ca2+ from the bath solution, or inclusion of EGTA (5 mM) in the patch pipette, also led to abolition of these currents, indicating that they were Ca2+ dependent, and that Ca2+ influx due to I(Ca,L) activated the currents. 4. When [Cl(‐)](O) or [Cl(‐)](i) was changed, the reversal potential (E(rev)) of the Ca2+‐activated currents shifted, thus behaving like a Cl(‐)‐selective ion channel as predicted by the Nernst equation. DIDS (1 mM) completely abolished the currents, also suggesting that they were I[Cl(Ca)]. 5. NKA (1 mu M) and caffeine (30 mM) transiently activated I[Cl(Ca)], and after that both agents markedly reduced I[Cl(Ca)] induced by I(Ca,L). This is probably due to sarcoplasmic reticulum (SR) Ca2+ release induced by NKA or caffeine, followed by inhibition of the Ca(2+)‐induced Ca2+ release from the SR. 6. The present results indicate that I[Cl(Ca)] can be activated by SR Ca2+ release due to NKA or caffeine (through IP(3) or ryanodine receptors) as well as by Ca2+ influx due to I(Ca,L). It also suggests that activation of I[Cl(Ca)] by NKA may be mediated by the production of IP(3), which releases Ca2+ from the SR.