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The aims of the present study were to evaluate the effects of puerarin on angiotensin II-induced cardiac fibroblast proliferation and to explore the molecular mechanisms of action. Considering the role of H 2 O 2 in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation, we hypothesized that modulating catalase activity would be a potential target in regulating the redox-sensitive pathways. Our results showed that the activation of Rac1 was dependent on the levels of intracellular H 2 O 2 . Puerarin blocked the phosphorylation of extracellular regulated protein kinases (ERK)1/2, abolished activator protein (AP)-1 binding activity, and eventually attenuated cardiac fibroblast proliferation through the inhibition of H 2 O 2 -dependent Rac1 activation. Further studies revealed that angiotensin II treatment resulted in decreased catalase protein expression and enzyme activity, which was disrupted by puerarin via the upregulation of catalase protein expression at the transcriptional level and the prolonged protein degradation. These findings indicated that the anti-proliferation mechanism of puerarin was mainly through blocking angiontensin II-triggered downregulation of catalase expression and H 2 O 2 -dependent Rac1 activation., (Copyright © 2018 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.)

Considering that α-1 repeat region may be involved in the ion binding and translocation of Na(+)-Ca(2+) exchanger (NCX), it is possible that the antibodies against NCX α-1 repeat may have a crucial action on NCX activity. The aim of the present study is to investigate the effect of antibody against α-1 repeat (117-137), designated as α-1(117-137), on NCX activity. The antibody against the synthesized α-1(117-137) was prepared and affinity-purified. Whole-cell patch clamp technique was used to study the change of Na(+)-Ca(2+) exchange current (I(Na/Ca)) in adult rat cardiomyocytes. To evaluate the functional specificity of this antibody, its effects on L-type Ca(2+) current (I(Ca,L)), voltage-gated Na(+) current (I(Na)) and delayed rectifier K(+) current (I(K)) were also observed. The amino acid sequences of α-1(117-137) in NCX and residues 1 076-1 096 within L-type Ca(2+) channel were compared using EMBOSS Pairwise Alignment Algorithms. The results showed that outward and inward I(Na/Ca) were decreased by the antibody against α-1(117-137) dose-dependently in the concentration range from 10 to 160 nmol/L, with IC(50) values of 18.9 nmol/L and 22.4 nmol/L, respectively. Meanwhile, the antibody also decreased I(Ca,L) in a concentration-dependent manner with IC(50) of 22.7 nmol/L. No obvious effects of the antibody on I(Na) and I(K) were observed. Moreover, comparison of the amino acid sequences showed there was 23.8% sequence similarity between NCX α-1(117-137) and residues 1 076-1 096 within L-type Ca(2+) channel. These results suggest that antibody against α-1(117-137) is a blocking antibody to NCX and can also decrease I(Ca,L) in a concentration-dependent manner, while it does not have obvious effects on I(Na) and I(K).

The present study was aimed to investigate the positive inotropic mechanism of carbachol (CCh) on rat ventricular myocytes. The effects of CCh on L-type calcium current (I(Ca,L)) and Na(+)/Ca(2+) exchange current (I(Na/Ca)) were investigated in isolated rat ventricular myocytes. After loading myocytes with Fura-2/AM, electrically triggered Ca(2+) transient and cell shortening in single myocyte were measured simultaneously using ion imaging system with charge-coupled device (CCD) camera. CCh (100 mumol/L) increased I(Na/Ca) in forward mode from (1.18 +/- 0.57) pA/pF in the control group to (1.65 +/- 0.52) pA/pF (P<0.01) and that in reverse mode from (1.11 +/- 0.49) pA/pF in the control group to (1.53 +/- 0.52) pA/pF (P<0.01), respectively. CCh had no effect on I(Ca,L). The stimulatory effect of CCh on I(Na/Ca) was blocked by application of atropine, a non-selective M muscarinic receptor antagonist, and methoctramine, a selective M(2) muscarinic receptor antagonist. CCh (100 mumol/L) increased cell shortening from (3.00 +/- 0.67) mum in the control group to (3.55 +/- 1.21) mum. Ca(2+) transient was also increased from 203.8 +/- 50.0 in the control group to 234.8 +/- 64.3 in 100 mumol/L CCh group. KB-R7943, a selective inhibitor of reverse mode Na(+)/Ca(2+) exchange, did not change the baseline level of cell shortening and Ca(2+) transient, while completely abolished CCh-induced increments of both Ca(2+) transient and cell shortening. CCh increased cell shortening and Ca(2+) transient in the presence of nicardipine, indicating that the positive inotropic effect of CCh was through activation of Na(+)/Ca(2+) exchange. Calcium sensitivity was not changed by CCh. Both atropine and methoctramine abolished the positive inotropic effects of CCh, demonstrating that CCh induced positive inotropism via the M(2) muscarinic receptor. The results suggest that CCh increases cell contraction and Ca(2+) transient in rat ventricular myocytes. This positive inotropic effect of CCh is through activation of reverse mode Na(+)/Ca(2+) exchange, and M(2) receptors are involved in mediating CCh-induced contraction.

Neuropeptide Y (NPY) co-exists with norepinephrine (NE) in sympathetic terminals, and is the most abundant neuropeptide in myocardium. Many studies have focused on the effects of NE on ion channels in cardiac myocytes and its physiological significance has been elucidated relatively profoundly. There have been few investigations, however, on the physiological significance of NPY in myocardium. The effects of NPY on L-type Ca2+ channel currents (I(Ca-L)) were evaluated in some studies and different results were presented, which might be attributed to the different species of animal tested and different methods used. It is necessary, therefore, to study the effects of NPY on ion channels in cardiac myocytes systematically and further to discuss the biological significance of their coexistence with NE in sympathetic terminals. The single ventricular myocytes from adult rat or guinea pig (only for measuring I(K)) were prepared using enzymatic dispersion. I(Ca-L), I(to), I(Na/Ca), I(Na) and I(K) in the cellular membrane were observed using whole cell voltage-clamp recording. In the present study, NPY from 1.0 to 100 nmol/L dose-dependently inhibited I(Ca-L) (P<0.01, n=5). The maximal rate of inhibition in this study reached 39% and IC(50) was 1.86 nmol/L. NPY had no effect on the voltage-dependence of calcium current amplitude and on the voltage-dependence of the steady-state gating variables. I(Ca-L) was activated at -30 mV, reaching the maximum at 0 mV. When both NE and NPY were applied with a concentration ratio of 500:1, 10 nmol/L NPY inhibited I(Ca-L) that had been increased by 5 mumol/L NE, which was consistent with the effect of NPY only on I(Ca-L). NPY also inhibited I(Na/Ca). At a concentration of 10 nmol/L, NPY inhibited inward and outward I(Na/Ca) from (0.27+/-0.11) pA/pF and (0.45+/-0.12) pA/pF to (0.06+/-0.01) pA/pF and (0.27+/-0.09) pA/pF, respectively (P<0.05, n=4). NPY at 10 nmol/L increased I(to) from (12.5+/-0.70) pA/pF to (14.7+/-0.59) pA/pF(P<0.05, n=4). NPY at 10 nmol/L did not affect I(Na) in rat myocytes and I(K) in guinea pig myocytes. NPY increased the speed of action potential depolarization and reduced action potential duration of I(Ca-L), I(Na/Ca) and I(to), which contributed to the reduction of contraction. These results indicate that the effects of NPY are opposite to the effects of NE on ion channels of cardiac myocytes.

Stimulation of cardiac mAChRs by carbachol (CCh) produces a biphasic inotropic response. The mechanisms of the positive inotropic response by higher concentration of CCh appear to be paradoxical. This article was aimed to study the mechanism of the positive inotropic effect of CCh in guinea pig ventricular myocytes. The effects of CCh on L-type calcium current (I(Ca)) and Na/Ca exchange current (I(Na/Ca)) were observed in voltage-clamped guinea pig ventricular myocytes by using Axon 200A amplifier. The results showed that CCh (100 micromol/L) increased both forward mode and reverse mode I(Na/Ca) from (1.2+/-0.1) pA/pF to (2.0+/-0.3) pA/pF for forward mode (P<0.01) and from (1.3+/-0.5) pA/pF to (2.1+/-0.8) pA/pF for reverse mode (P<0.01), respectively. CCh had no effect on I(Ca). The stimulating effect of CCh on I(Na/Ca) could be blocked by application of atropine, a nonselective blocker of muscarinic receptors, which means that the stimulating effect of CCh is through the activation of muscarinic receptors. We made a further study by using methoctramine, a selective antagonist of M2 muscarinic receptors. It completely abolished I(Na/Ca) induced by 100 micromol/L CCh, indicating that the effect of CCh on I(Na/Ca) was mediated by M2 muscarinic receptors. It is generally accepted that contraction in cardiac myocytes results from elevation of intracellular Ca2+ concentration. Ca2+ enters the cells through two pathways: L-type Ca2+ channels and, less importantly, reverse mode Na/Ca exchange. The calcium influx via both pathways promotes the contraction of cardiac myocytes. Because CCh had no effect on L-type Ca2+ current, the increase in Na/Ca exchange current might be the main factor in the positive inotropism of CCh. These results suggest that the positive inotropic effect of CCh in guinea pig heart is through stimulation of Na/Ca exchange and is mediated by M2 muscarinic receptors.

To study the inotropic effect of enhanced Na(+)-Ca(2+) exchange in the rat papillary muscles and isolated heart, the developed tension in the rat papillary muscles was measured and the left ventricular functions were assessed in the isolated rat heart. E-4031, a selective activator for Na(+)-Ca(2+) exchange in rats, concentration-dependently increased the developed contractile tension in the rat papillary muscles (P<0.05, n=6) and the left ventricular functions in the isolated heart; KB-R7943, a selective Na(+)-Ca(2+) exchange inhibitor, exhibited opposite effect. A combination of 0.5 micromol/L ouabain and 3.0 micromol/L E-4031 resulted in a potentiation of the developed contractile tension of the rat papillary muscles from 0.25+/-0.03 g to 0.29+/-0.04 g. The combination also enhanced the augmentation of the left ventricular functions induced by ouabain. These results indicate that E-4031 exerts a positive inotropic effect on the rat papillary muscles and isolated heart via increasing the activity of Na(+)-Ca(2+) exchange, and potentiates the positive inotropic effects of ouabain.

Calcium sensitizers exert positive inotropic effects without increasing intracellular Ca(2+). Thus, they avoid the undesired effects of Ca(2+) overload such as arrhythmias and cell injury, but most of them may impair myocyte relaxation. However, MCI-154, also a calcium sensitizer, has no impairment to cardiomyocyte relaxation. To clarify the underlying mechanisms, we examined the effects of MCI-154 on Ca(2+) transient and cell contraction using ion imaging system, and its influence on L-type Ca(2+) current and Na(+)/ Ca(2+) exchange current with patch clamp technique in rat ventricular myocytes as well. The results showed that: (1) MCI-154 (1-100 micromol/L) had no effect on L-type Ca(2+) current; (2) MCI-154 concentration-dependently increased cell shortening from 5.00+/-1.6 microm of control to 6.2+/-1.6 microm at 1 micromol/L, 8.7+/-1.6 microm at 10 micromol/L and 14.0+/-1.4 microm at 100 micromol/L, respectively, with a slight increase in Ca(2+) transient amplitude and an abbreviation of Ca(2+) transient restore kinetics assessed by time to 50% restore (TR(50)) and time to 90% restore (TR(90)); (3) MCI-154 dose-dependently increased the electrogenic Na(+)/ Ca(2+) exchange current both in the inward and the outward directions in rat ventricular myocytes. These results indicate that MCI-154 exerted a positive inotropic action without impairing myocyte relaxation. The stimulation of inward Na(+)/ Ca(2+) exchange current may accelerate the Ca(2+) efflux, leading to abbreviations of TR(50) and TR(90) in rat myocytes. The findings suggest that the improvement by MCI-154 of myocyte relaxation is attributed to the forward mode of Na(+)/ Ca(2+) exchange.

The effects of 5-(N,N-dimethyl)amiloride (DMA) (a blocker of Na(+)/H(+) exchanger or Na(+)/Ca(2+) exchanger) on calcium transient and cell contraction in isolated ventricular myocytes in normal rats and rats with myocardial hypertrophy were examined using ion imaging system with a charge coupled digital camera (CCD camera). Loading myocytes with Fura-2, electrically triggered Ca(2+) transients and cell shortening were measured simultaneously. The results showed that 10 micromol/L DMA increased Ca(2+) transient and cell shortening from 209.60+/-54.96 and 3.07+/-0.97 micrometer to 238.50+/-80.41 and 4.07+/-1.02 micrometer, respectively (P<0.05), which was completely abolished by application of KB-R7943, a specific reverse mode Na(+)/Ca(2+) exchanger blocker. After blocking L-type Ca(2+) channels by nicardipine, DMA also enhanced Ca(2+) transient and cell shortening. In rats with myocardial hypertrophy, DMA showed the common pharmacologic profile as in normal rats but more intense stimulating effects on Ca(2+) transient and cell contraction. The results suggest that DMA increase Ca(2+) transient and cell contraction via stimulating reverse mode Na(+)/ Ca(2+) exchange, and the stimulating effect is more pronounced in rats with myocardial hypertrophy than in normal ones.