Angiotensin-(1-7) increases neuronal potassium current via a nitric oxide-dependent mechanism.

Actions of angiotensin-(1-7) [Ang-(1-7)], a heptapeptide of the renin-angiotensin system, in the periphery are mediated, at least in part, by activation of nitric oxide (NO) synthase (NOS) and generation NO(·). Studies of the central nervous system have shown that NO(·) acts as a sympathoinhibitory molecule and thus may play a protective role in neurocardiovascular diseases associated with sympathoexcitation, such as hypertension and heart failure. However, the contribution of NO in the intraneuronal signaling pathway of Ang-(1-7) and the subsequent modulation of neuronal activity remains unclear. Here, we tested the hypothesis that neuronal NOS (nNOS)-derived NO(·) mediates changes in neuronal activity following Ang-(1-7) stimulation. For these studies, we used differentiated catecholaminergic (CATH.a) neurons, which we show express the Ang-(1-7) receptor (Mas R) and nNOS. Stimulation of CATH.a neurons with Ang-(1-7) (100 nM) increased intracellular NO levels, as measured by 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM) fluorescence and confocal microscopy. This response was significantly attenuated in neurons pretreated with the Mas R antagonist (A-779), a nonspecific NOS inhibitor (nitro-L-arginine methyl ester), or an nNOS inhibitor (S-methyl-L-thiocitrulline, SMTC), but not by endothelial NOS (eNOS) or inhibitory NOS (iNOS) inhibition {L-N-5-(1-iminoethyl)ornithine (L-NIO) and 1400W, respectively}. To examine the effect of Ang-(1-7)-NO(·) signaling on neuronal activity, we recorded voltage-gated outward K(+) current (I(Kv)) in CATH.a neurons using the whole cell configuration of the patch-clamp technique. Ang-(1-7) significantly increased I(Kv), and this response was inhibited by A-779 or S-methyl-L-thiocitrulline, but not L-NIO or 1400W. These findings indicate that Ang-(1-7) is capable of increasing nNOS-derived NO(·) levels, which in turn, activates hyperpolarizing I(Kv) in catecholaminergic neurons.

The attenuation of central angiotensin II-dependent pressor response and intra-neuronal signaling by intracarotid injection of nanoformulated copper/zinc superoxide dismutase.

Adenoviral-mediated overexpression of the intracellular superoxide (O(2)(*-)) scavenging enzyme copper/zinc superoxide dismutase (CuZnSOD) in the brain attenuates central angiotensin II (AngII)-induced cardiovascular responses. However, the therapeutic potential for adenoviral vectors is weakened by toxicity and the inability of adenoviral vectors to target the brain following peripheral administration. Therefore, we developed a non-viral delivery system in which CuZnSOD protein is electrostatically bound to a synthetic poly(ethyleneimine)-poly(ethyleneglycol) (PEI-PEG) polymer to form a polyion complex (CuZnSOD nanozyme). We hypothesized that PEI-PEG polymer increases transport of functional CuZnSOD to neurons, which inhibits AngII intra-neuronal signaling. The AngII-induced increase in O(2)(*-), as measured by dihydroethidium fluorescence and electron paramagnetic resonance spectroscopy, was significantly inhibited in CuZnSOD nanozyme-treated neurons compared to free CuZnSOD- and non-treated neurons. CuZnSOD nanozyme also attenuated the AngII-induced inhibition of K(+) current in neurons. Intracarotid injection of CuZnSOD nanozyme into rabbits significantly inhibited the pressor response of intracerebroventricular-delivered AngII; however, intracarotid injection of free CuZnSOD or PEI-PEG polymer alone failed to inhibit this response. Importantly, neither the PEI-PEG polymer alone nor the CuZnSOD nanozyme induced neuronal toxicity. These findings indicate that CuZnSOD nanozyme inhibits AngII intra-neuronal signaling in vitro and in vivo.

Mitochondria-produced superoxide mediates angiotensin II-induced inhibition of neuronal potassium current.

Reactive oxygen species (ROS), particularly superoxide (O(2)(.-)), have been identified as key signaling intermediates in ANG II-induced neuronal activation and sympathoexcitation associated with cardiovascular diseases, such as hypertension and heart failure. Studies of the central nervous system have identified NADPH oxidase as a primary source of O(2)(.-) in ANG II-stimulated neurons; however, additional sources of O(2)(.-), including mitochondria, have been mostly overlooked. Here, we tested the hypothesis that ANG II increases mitochondria-produced O(2)(.-) in neurons and that increased scavenging of mitochondria-produced O(2)(.-) attenuates ANG II-dependent intraneuronal signaling. Stimulation of catecholaminergic (CATH.a) neurons with ANG II (100 nM) increased mitochondria-localized O(2)(.-) levels, as measured by MitoSOX Red fluorescence. This response was significantly attenuated in neurons overexpressing the mitochondria-targeted O(2)(.-)-scavenging enzyme Mn-SOD. To examine the biological significance of the ANG II-mediated increase in mitochondria-produced O(2)(.-), we used the whole cell configuration of the patch-clamp technique to record the well-characterized ANG II-induced inhibition of voltage-gated K(+) current (I(Kv)) in neurons. Adenovirus-mediated Mn-SOD overexpression or pretreatment with the cell-permeable antioxidant tempol (1 mM) significantly attenuated ANG II-induced inhibition of I(Kv). In contrast, pretreatment with extracellular SOD protein (400 U/ml) had no effect. Mn-SOD overexpression also inhibited ANG II-induced activation of Ca(2+)/calmodulin kinase II, a redox-sensitive protein known to modulate I(Kv). These data indicate that ANG II increases mitochondrial O(2)(.-), which mediates, at least in part, ANG II-induced activation of Ca(2+)/calmodulin kinase II and inhibition of I(Kv) in neurons.

Effects of strophanthidin on intracellular calcium concentration in ventricular myocytes of guinea pig.

Effect of strophanthidin (Str) on intracellular calcium concentration ([Ca2+]i) was investigated on isolated ventricular myocytes of guinea pig. Single ventricular myocytes were obtained by enzymatic dissociation technique. Fluorescent signal of [Ca2+]i was detected with confocal microscopy after incubation of cardiomycytes in Tyrode' s solution with Fluo3-AM. The result showed that Str increased [Ca2+]i in a concentration-dependent manner. The ventricular myocytes began to round-up into a contracture state once the peak level of [Ca2+]i was achieved in the presence of Str (10 micromol L(- 1)), but remained no change in the presence of Str (1 and 100 nmol L(-1)). Tetrodotoxin (TTX), nisodipine, and high concentration of extracellular Ca2+ changed the response of cardiomycytes to Str (1 and 100 nmol L(-1)) , but had no obvious effects on the action of Str (10 micromol L(-1)). The elevation of [Ca2+]i caused by Str at all of the detected concentrations was partially antagonized by rynodine (10 micromol L(-1)) or the removal of Ca2+ from Tyrode's solution. In Na+, K+ -free Tyrode' s solution, the response of cardiomycytes in [Ca2+]i elevation to Str (10 micromol L(-1)) was attenuated, while remained no change to Str (1 and 100 nmol L(-1)). TTX, nisodipine, and high concentration of extracellular Ca2+ changed the response of cardiomycytes to Str at all of the detected concentrations in Na+, K+ -free Tyrode's solution. The study suggests that the elevation of [Ca2+]i by Str at the low (nomomolar) concentrations is partially mediated by the extracellular calcium influx through Ca2+ channel or a "slip mode conductance" of TTX sensitive Na+ channel. While the effect of Str at high (micromolar) concentrations was mainly due to the inhibition of Na+, K+ -ATPase. Directly triggering the release of intracellular Ca2+ from sarcoplasmic reticulum (SR) by Str may be also involved in the mechanism of [Ca2+]i elevation.

Effect of resveratrol on L-type calcium current in rat ventricular myocytes.

AIM: To study the effect of resveratrol on L-type calcium current (I(Ca-L)) in isolated rat ventricular myocytes and the mechanisms underlying these effects. METHODS: I(Ca-L) was examined in isolated single rat ventricular myocytes by using the whole cell patch-clamp recording technique. RESULTS: Resveratrol (10-40 micromol/L) reduced the peak amplitude of I(Ca-L) and shifted the current-voltage (I-V) curve upwards in a concentration-dependent manner. Resveratrol (10, 20, 40 micromol/L) decreased the peak amplitude of I(Ca-L) from -14.2+/-1.5 pA/pF to -10.5+/-1.5 pA/pF (P<0.05), -7.5+/-2.4 pA/pF (P<0.01), and -5.2+/-1.2 pA/pF (P<0.01), respectively. Resveratrol (40 micromol/L) shifted the steady-state activation curve of I(Ca-L) to the right and changed the half-activation potential (V0.5) from -19.4+/-0.4 mV to -15.4+/-1.9 mV (P<0.05). Resveratrol at a concentration of 40 micromol/L did not affect the steady-state inactivation curve of I(Ca-L), but did markedly shift the time-dependent recovery curve of I(Ca-L) to the right, and slow down the recovery of I(Ca-L) from inactivation. Sodium orthovanadate (Na(3)VO(4); 1 mmol/L), a potent inhibitor of tyrosine phosphatase, significantly inhibited the effects of resveratrol (P<0.01). CONCLUSION: Resveratrol inhibited I(Ca-L) mainly by inhibiting the activation of L-type calcium channels and slowing down the recovery of L-type calcium channels from inactivation. This inhibitory effect of resveratrol was mediated by the inhibition of protein tyrosine kinase in rat ventricular myocytes.

[Adenosine reduces intracellular free calcium concentration in guinea pig ventricular myocytes].

AIM: To observe the effects of adenosine on intracellular calcium concentration ([Ca2+]i) level in guinea pig ventricular myocytes and to define the possible mechanisms involved. METHODS: The effects of adenosine on [Ca2+]i were investigated in guinea pig ventricular myocytes. [Ca2+]i was detected by laser confocal microscopy and represented by relative fluorescent intensity ((FI-FI0)/FI0, %, FIo: control, FI: administration of drugs). RESULTS: (1) Adenosine (10, 50, 100 micromol/L) reduced [Ca2+]i of ventricular myocytes in both normal Tyrode's solution and Ca(2+) -free Tyrode's solution in a concentration-dependent manner. (2) Tyrode's solution containing 30 mmol/L KCl (high K+ Tyrode's solution) induced [Ca2+]i elevation in ventricular myocytes, while adenosine (10, 50, 100 micromol/L) markedly inhibited the increase in [Ca2+]i induced by KCl. (3) Pretreatment with DPCPX (1 micromol/L) significantly reduced the effects of adenosine (100 micromol/L) in high K+ Tyrode's solution. The effects of adenosine (100 micromol/L) on [Ca2+]i in high K+ Tyrode's solution were also partially attenuated by pretreatment with L-NAME (1 mmol/L). (4) Adenosine (100 micromol/L) markedly inhibited the low concentration of ryanodine-induced [Ca2+]i increase in Ca(2+) -free Tyrode's solution. (5) When the propagating waves of elevated [Ca2+]i (Ca2+ waves) were produced by increasing extracellular Ca2+ concentration from 1 mmol/L to 10 mmol/L, adenosine (100 micromol/L) could block the propagating waves of elevated [Ca2+]i, reduce the frequency and duration of propagating waves, and reduce [Ca2+]i as well. CONCLUSION: Adenosine may reduce the [Ca2+]i in isolated guinea pig ventricular myocytes via inhibiting Ca2+ influx and alleviating Ca2+ release from sarcoplasmic reticulum(SR). The reduction of Ca2+ influx might be due to the inhibition of voltage-dependent Ca2+ channel via adenosine A1 receptor, and NO might be involved in this process.

Effect of genistein on L-type calcium current in guinea pig ventricular myocytes.

This paper was aimed to study the effect of genistein (GST) on L-type calcium current (I(Ca,L)) in isolated guinea pig ventricular myocytes using whole cell patch-clamp recording technique. The results are as follows. (1) GST (10, 50, 100 micromol/L) reduced the voltage-activated peak amplitude of I(Ca,L) in a concentration-dependent manner. Daidzein (100 micromol/L), a structural analogue of GST which has little or no inhibitory effect on tyrosine kinase, produced no effect over the same concentration range on I(Ca,L) (n=5, P>0.05). (2) GST up- shifted the current-voltage (I-V) curve, but the characteristics of I-V relationship were not significantly altered, and the maximal activation voltage of I(Ca,L) was not different from that of control. GST did not affect the activation kinetics of I(Ca,L). (3) GST markedly shifted the steady-state inactivation curve of I(Ca,L) to the left, and accelerated the voltage-dependent steady-state inactivation of I(Ca,L). V(0.5) value was -28.6 +/-0.6 mV in the control and -32.8 +/-1.1 mV in the presence of GST. The kappa values were 5.8 +/-0.5 mV and 6.5 +/-0.9 mV, respectively (n=6, P<0.05). (4) GST markedly shifted the curve of time-dependent recovery of I(Ca,L) from the steady-state inactivation to the right, and slowed down the recovery of I(Ca,L) from inactivation (n=7, P<0.01). (5) Sodium orthovanadate (1 mmol/L), a potent inhibitor of tyrosine phosphatase, significantly inhibited GST-induced inhibition (n=6, P<0.01). From the results obtained it is concluded that genistein inhibits I(Ca,L) and acts on the inactivated state of L-type calcium channel. This inhibitory effect of GST involves protein tyrosine kinase inhibition in guinea pig ventricular myocytes.

Effects of low-dose capsaicin on L-type calcium current in guinea pig ventricular myocytes.

The purpose of this study was to examine the effects of low-dose capsaicin (CAP) on L-type calcium current (I(Ca-L) ) in guinea pig ventricular myocytes and the underlying mechanism. I(Ca-L) was examined in isolated single guinea pig ventricular myocytes by using whole-cell patch clamp technique. CAP (1-25 nmol/L) increased the voltage-dependently activated peak amplitude of I(Ca-L) and downshifted the current-voltage (I-V) curve. CAP (1, 10, 25 nmol/L) increased the peak amplitude of I(Ca-L) from -9.67+/-0.7 pA/pF to -10.21+/-0.8 pA/pF (P>0.05), to -11.37+/-0.8 pA/pF and to -12.84+/-0.9 pA/pF (P<0.05), respectively. CAP 25 nmol/L shifted the steady-state activation curve of I(Ca-L) to the left and changed half activation potential (V(0.5)) from (-20.76+/-2.0) mV to (-26.71+/-3.0) mV (P<0.05), indicating that low-dose CAP may modify the voltage-dependent activation of calcium channel. Low-dose of CAP did not affect the steady-state inactivation curve of I(Ca-L) or half-recovery time of Ca(2+) channel from inactivation. Ruthenium red (RR, 10 micromol/L), a vanilloid receptor (VR1) blocker, antagonized the effects of low-dose CAP. These results suggest that low-dose CAP increases I(Ca-L) mainly by shifting its steady-state activation curve to the left. Such effects may be mediated by VR1.

Cholecystokinin octapeptide increases free intracellular calcium of guinea pig cardiomyocytes through activation of Ca2+ channel and tyrosine kinase.

The aim of the present study was to explore the effect of cholecystokinin octapeptide (CCK-8) on [Ca(2+)](i) and its signal transduction mechanism in isolated guinea pig cardiomyocytes. [Ca(2+)](i) was measured by laser scanning confocal microscopy in single ventricular myocytes which were dissociated by enzymatic dissociation method and loaded with Fluo 3-AM. The changes in [Ca(2+)](i) were represented by fluorescent intensity (F(i)) or relative fluorescent intensity (F(i)/F(O)%). The results obtained are as follows. (1) In the normal Tyrode's solution containing 1.0 mmol/ L Ca(2+), CCK-8 (1-10(4) pmol/L) elicited a rapid and marked increase in [Ca(2+)](i). (2) When cardiomyocytes were pretreated with the Ca(2+) chelator EGTA (3 mmol/L) and Ca(2+) channel antagonist nisoldipine (0.5 micromol/L) for 5 min, CCK-8 (10(2)pmol/L) caused a slow and small increase in [Ca(2+)](i) (p< 0.01). (3) Pretreatment with the nonselected CCK- receptor (CCK-R) antagonist proglumide (6 micromol/L) or the tyrosine kinase inhibitor genistein (1 micromol/L) for 5 min could inhibit the increase of [Ca(2+)](i) induced by CCK-8 (10(2) pmol/L) (p<0.01). The results suggest that CCK-8 increases the [Ca(2+)](i) via activating the receptor-operated Ca(2+) channel and eliciting the influx of Ca(2+) in isolated guinea pig cardiomyocytes, in which tyrosine kinase may be involved.

Relationship between cardiotonic effects and inhibition on cardiac sarcolemmal Na+,K+-ATPase of strophan-thidin at low concentrations.

AIM: To evaluate the effects of strophanthidin (Str) on cardiac contractile function and sarcolemmal Na+, K+-ATPase activities in isolated guinea-pig hearts. METHODS: Isolated guinea-pig hearts were perfused through aorta in a Langendorff mode. Heart rate (HR), left ventricular pressure (LVP), and first derivatives (+/-dp/dt(max)) of LVP were recorded by eight-channel physiological instrument. Cardiac sarcolemmal Na+, K+-ATPase activities were determined with colorimetry. RESULTS: Str 0.1 nmol/L stimulated the Na+, K+-ATPase activities (P<0.05), but had no effect on HR, LVP, and +/-dp/dt(max). Str 1 nmol/L increased +dp/dt(max) (P<0.05) and Na+, K+-ATPase activities (P<0.01). Str 10 and 100 nmol/L significantly increased both LVP (P<0.05) and +dp/dt(max) (P<0.05 or P<0.01), and had no significant effects on Na+, K+-ATPase activities. However, Str 1-100 micromol/L at first enhanced the LVP and +dp/dtmax (P<0.01), then reduced them resulting from irregular contraction, and effects of Str on Na+, K+-ATPase activities revealed a concentration-dependent inhibition (P<0.01). CONCLUSION: The positive inotropic effects and irregular contraction produced by Str at higher concentrations result from the inhibition of Na+, K+-ATPase activities, and the positive inotropic effects of Str at lower concentrations are not related to the inhibition of the Na+, K+-ATPase activities.

Effect of agmatine on intracellular free calcium concentration in isolated rat ventricular myocytes.

The present study was to investigate the effects of agmatine (Agm) on free intracellular calcium concentration ([Ca(2+)]( i )) of isolated rat ventricular myocytes. [Ca(2+)]( i ) was measured by confocal microscopy in single rat ventricular myocytes which were dissociated by enzymatic dissociation method and loaded with Fluo 3-AM. The changes in [Ca(2+)]( i ) were represented by fluorescence intensity (FI) or relative fluorescence intensity (F/F(0)%). The results showed that the control level of FI value of single rat ventricular myocytes was 128.8+/-13.8 and 119.6+/-13.6 in the presence of normal Tyrode's solution containing Ca(2+) 1.0 mmol/L and Ca(2+)-free Tyrode's solution, respectively. There was no difference between these two groups (P>0.05). Agm 0.1, 1, and 10 mmol/L significantly reduced the [Ca(2+)]( i ) in both extracellular solutions in a concentration-dependent manner. The similar effect of Agm on [Ca(2+)]( i ) was also observed in the presence of EGTA 3 mmol/L. KCl 60 mmol/L, PE 30 micromol/L, and Bay-K-8644 10 micromol/L, all these substances induced [Ca(2+)]( i ) elevations in ventricular myocytes. Agm (0.1, 1, and 10 mmol/L) markedly inhibited the increase in [Ca(2+)]( i ) induced by KCl, phenylephrine (PE), and Bay-K-8644. When Ca(2+) waves were produced by increasing extracellular Ca(2+) concentration from 1 to 10 mmol/L, 1 mmol/L Agm could block the propagating waves of elevated [Ca(2+)]( i ), and reduce the velocity and duration of propagating waves. These results suggest that Agm possesses an inhibitory effects on [Ca(2+)]( i ) via blocking voltage-dependent Ca(2+) channel, and possibly by alleviating calcium release from SR in single isolated rat ventricular myocytes.

Effects of low concentration of dihydroouabain on intracellular calcium in guinea pig ventricular myocytes.

The effects of low concentration of dihydroouabain (DHO) on intracellular calcium concentration ([Ca(2+)](i)) were investigated in guinea pig ventricular myocytes. [Ca(2+)](i) was detected by confocal microscopy and represented by fluorescent intensity. DHO (1 fmol/L~1 mmol/L) increased [Ca(2+)](i), especially at 10 pmol/L. Nisoldipine, egtazic acid, or tetrodotoxin partially inhibited the effect of 10 pmol/L DHO on [Ca(2+)](i). The effects of DHO remained in the absence of extracellular K(+) and Na(+). These results suggest that low concentration of DHO might increase [Ca(2+)](i) via the receptor-operated Ca(2+) channels, TTX-sensitive Na(+) channels or/and triggering of intracellular calcium release; Na(+)/K(+) pump and Na(+)/Ca(2+) exchange seem not involved in the effect of DHO.

Effect of agmatine on L-type calcium current in rat ventricular myocytes.

AIM: To study the effect of agmatine (Agm) on L-type calcium current (I(Ca-L)) in rat ventricular myocytes. METHODS: Whole-cell configuration of the patch-clamp technique was used to record I(Ca-L) in single rat ventricular myocytes which were dissociated by enzymatic dissociation method. RESULTS: (1) Agm (0.5, 1, 2 mmol/L) reduced the voltage-dependently activated peak amplitude of I(Ca-L) (pA) from 1451+/-236 (control) to 937+/-105 (n=8, P <0.05), 585+/-74 (n=8, P <0.01), and to 301+/-156 (n=8, P <0.01) in a concentration-dependent manner. (2) Agm (1 mmol/L) blocked I(Ca-L) in a use-dependent manner. The degree of use-dependent blocking effect was 53 %+/-12 % (n=8, P <0.05) at 1 Hz, and 69 %+/-11 % (n=8, P <0.01) at 3 Hz. (3) Agm upshifted the current-voltage (I-V) curve, but the characteristics of I-V relationship were not significantly altered by Agm, the maximal activation voltage of I(Ca-L) was not different from that of control. Steady-state activation of I(Ca-L) was not affected markedly. The half activation potential (V(0.5)) and the slope factor (k) were not significantly different from those of the control. V(0.5) value was (-20.2+/-2.5) mV in the control and (-20.5+/-2.7) mV in the presence of Agm 1 mmol/L. The k value was (7.1+/-0.4) mV and (7.5+/-0.5) mV, respectively (n=8, P >0.05). (4) Agm 1 mmol/L markedly shifted the steady-state inactivation curve of I(Ca-L) to the left, and accelerated the voltage-dependent steady-state inactivation of calcium current. V(0.5) value was (-32+/-6) mV in the control and (-40+/-5) mV in the presence of Agm. The k value was (7.6+/-0.9) mV and (12.5+/-1.1) mV, respectively (n=8, P <0.05). (5) Agm 1 mmol/L markedly delayed half-recovery time of Ca2+ channel from inactivation (92+/-28) ms to (249+/-26) ms (n=8, P <0.01). CONCLUSION: Agm inhibited I(Ca-L) and mainly acted on the inactivated state of L-type calcium channel, manifested as acceleration of calcium channel inactivation and slowdown of recovery from inactivated state in rat ventricular myocytes.