The beneficial health effects of flavonoids on the cardiovascular system: Focus on K+ channels.

Substantial experimental evidences support the hypothesis that dietary flavonoid intake has a favourable impact on cardiovascular diseases such as systemic, arterial hypertension and coronary artery diseases, which represent the leading cause of morbidity and mortality worldwide. The biological effects of flavonoids involve complex biochemical interactions with numerous, specific, cellular and molecular targets. K+ channels, fine modulators of both cardiac action potential and vascular cell membrane potential, represent one of these targets. Overexpression, downregulation or dysfunction of these channel proteins are the cause of many cardiovascular diseases. Therefore, it appears of particular interest a detailed analysis of the flavonoid potential, direct/indirect modulation of cardiovascular K+ channels as these natural compounds ingested with the diet, despite extensive gut metabolism, may accumulate at cellular level in the form of the parent aglycones. The present review will portray their effects on cardiovascular K+ channels. Molecular docking was used to strengthen experimental evidences and describe flavonoid-channel interactions at molecular level.

Cancer cell permeability-glycoprotein as a target of MDR reverters: possible role of novel dihydropyridine derivatives.

The overexpression of permeability-glycoprotein (P-gp) and other drug transporters (ATP-binding cassette) confers a multidrug resistance (MDR) phenotype on cells in various diseases, including many forms of cancer. Development of MDR is one of the main reasons of failure in malignant tumour chemotherapy, as tumour cells, by increasing drug efflux, acquire cross-resistance to many structurally and functionally unrelated anticancer agents, which therefore never achieve effective intracellular concentrations. Endeavouring to find MDR-reverters is a crucial task for exploring new anti-cancer therapeutic intervention. Although many P-gp inhibitors have so far been identified, it is widely recognised that their interaction with P-gp is a complex process and, presently, the details of the mechanisms of action are still a matter of debate. These compounds turned out, however, to be of limited clinical usefulness owing to their inherent pharmacological activities (first generation compounds) and their accessory, inhibiting activity on CYP enzyme system (second generation compounds). Moreover, recent advances of the knowledge on P-gp structure and function and on the mechanisms of P-gp inhibition will prove fruitful for the development of novel therapeutically effective P-gp inhibitors. A dibenzoyl-1,4-dihydropyridine compound (DP7) has been shown to be a powerful P-gp inhibitor, almost devoid of cardiovascular effects, but capable of inhibiting liver CYP3A. DP7 is considered a lead compound for the development of novel dihydropyridines which do not affect CYP enzyme system but still retain the activity towards ABC-efflux transporters.

(+/-)-Naringenin as large conductance Ca(2+)-activated K+ (BKCa) channel opener in vascular smooth muscle cells.

UNLABELLED: BACKGROUND AND PURPOSE. The aim of this study was to investigate, in vascular smooth muscle cells, the mechanical and electrophysiological effects of (+/-)-naringenin. EXPERIMENTAL APPROACH: Aorta ring preparations and single tail artery myocytes were employed for functional and patch-clamp experiments, respectively. KEY RESULTS: (+/-)-Naringenin induced concentration-dependent relaxation in endothelium-denuded rat aortic rings pre-contracted with either 20 mM KCl or noradrenaline (pIC(50) values of 4.74 and 4.68, respectively). Tetraethylammonium, iberiotoxin, 4-aminopyridine and 60 mM KCl antagonised (+/-)-naringenin-induced vasorelaxation, while glibenclamide did not produce any significant antagonism. Naringin [(+/-)-naringenin 7-beta-neohesperidoside] caused a concentration-dependent relaxation of rings pre-contracted with 20 mM KCl, although its potency and efficacy were significantly lower than those of (+/-)-naringenin. In rat tail artery myocytes, (+/-)-naringenin increased large conductance Ca(2+)-activated K(+) (BK(Ca)) currents in a concentration-dependent manner; this stimulation was iberiotoxin-sensitive and fully reversible upon drug wash-out. (+/-)-Naringenin accelerated the activation kinetics of BK(Ca) current, shifted, by 22 mV, the voltage dependence of the activation curve to more negative potentials, and decreased the slope of activation. (+/-)-Naringenin-induced stimulation of BK(Ca) current was insensitive either to changes in the intracellular Ca(2+) concentration or to the presence, in the pipette solution, of the fast Ca(2+) chelator BAPTA. However, such stimulation was diminished when the K(+) gradient across the membrane was reduced. CONCLUSIONS AND IMPLICATIONS: The vasorelaxant effect of the naturally-occurring flavonoid (+/-)-naringenin on endothelium-denuded vessels was due to the activation of BK(Ca) channels in myocytes.

Functional, electrophysiological and molecular docking analysis of the modulation of Cav 1.2 channels in rat vascular myocytes by murrayafoline A.

BACKGROUND AND PURPOSE: The carbazole alkaloid murrayafoline A (MuA) enhances contractility and the Ca(2+) currents carried by the Cav 1.2 channels [ICa1.2 ] of rat cardiomyocytes. As only few drugs stimulate ICa1.2 , this study was designed to analyse the effects of MuA on vascular Cav 1.2 channels. EXPERIMENTAL APPROACH: Vascular activity was assessed on rat aorta rings mounted in organ baths. Cav 1.2 Ba(2+) current [IBa1.2 ] was recorded in single rat aorta and tail artery myocytes by the patch-clamp technique. Docking at a 3D model of the rat, α1c central pore subunit of the Cav 1.2 channel was simulated in silico. KEY RESULTS: In rat aorta rings MuA, at concentrations ≤14.2 µM, increased 30 mM K(+) -induced tone and shifted the concentration-response curve to K(+) to the left. Conversely, at concentrations >14.2 µM, it relaxed high K(+) depolarized rings and antagonized Bay K 8644-induced contraction. In single myocytes, MuA stimulated IBa1.2 in a concentration-dependent, bell-shaped manner; stimulation was stable, incompletely reversible upon drug washout and accompanied by a leftward shift of the voltage-dependent activation curve. MuA docked at the α1C subunit central pore differently from nifedipine and Bay K 8644, although apparently interacting with the same amino acids of the pocket. Neither Bay K 8644-induced stimulation nor nifedipine-induced block of IBa1.2 was modified by MuA. CONCLUSIONS AND IMPLICATIONS: Murrayafoline A is a naturally occurring vasoactive agent able to modulate Cav 1.2 channels and dock at the α1C subunit central pore in a manner that differed from that of dihydropyridines.

In vitro and in silico analysis of the vascular effects of asymmetrical N,N-bis(alkanol)amine aryl esters, novel multidrug resistance-reverting agents.

Asymmetrical N,N-bis(alkanol)amine aryl esters (FRA77, GDE6, and GDE19) are potent multidrug resistance (MDR) reversers. Their structures loosely remind that of the Ca(2+) antagonist verapamil. Therefore, the aim of this study was to investigate their vascular activity in vitro. Their effects on the mechanical activity of fresh and cultured rat aorta rings on Cav1.2 channel current (I Ca1.2) of A7r5 cells and their cytotoxicity on A7r5 and EA.hy926 cells were analyzed. Docking at the rat α1C subunit of the Cav1.2 channel was simulated in silico. Compounds tested were cytotoxic at concentrations >1 µM (FRA77, GDE6, GDE19) and >10 µM (verapamil) in EA.hy926 cells, or >10 µM (FRA77, GDE6, GDE19) and at 100 µM (verapamil) in A7r5 cells. In fresh rings, the three compounds partly antagonized phenylephrine and 60 mM K(+) (K60)-induced contraction at concentrations ≥1 and ≥3 µM, respectively. On the contrary, verapamil fully relaxed rings pre-contracted with both agents. In cultured rings, 10 µM GDE6, GDE19, FRA77, and verapamil significantly reduced the contractile response to both phenylephrine and K60. Similarly to verapamil, the three compounds docked at the α1C subunit, interacting with the same amino acids residues. FRA77, GDE6, and GDE19 inhibited I Ca1.2 with IC50 values 1 order of magnitude higher than that of verapamil. FRA77-, GDE6-, and GDE19-induced vascular effects occurred at concentrations that are at least 1 order of magnitude higher than those effectively reverting MDR. Though an unambiguous divergence between MDR reverting and vascular activity is of overwhelming importance, these findings consistently contribute to the design and synthesis of novel and potent chemosensitizers.

Effects of some sterically hindered phenols on whole-cell Ca(2+) current of guinea-pig gastric fundus smooth muscle cells.

1. The aim of the present study was to investigate the effects of extracellular application of some sterically-hindered phenols, namely 3-t-butyl-4-hydroxyanisole (BHA), 3,5-di-t-butyl-4-hydroxyanisole (DTBHA) and the dimer of BHA, 2,2'-dihydroxy-3,3'-di-t-butyl-5,5'-dimethoxydiphenyl (DIBHA), on the whole-cell Ca(2+) current (I(Ca)) of freshly isolated smooth muscle cells from the guinea-pig gastric fundus, in the presence of a range of Ca(2+) concentrations (1 -- 5 mM) using the patch-clamp technique. The influx of Ca(2+) had characteristics of L-type I(Ca) (I(Ca(L))). 2. BHA as well as DTBHA inhibited I(Ca(L)) in a concentration-dependent manner, during depolarization to 10 mV from a holding potential of -50 mV. Bath application of BHA (50 microM) and DTBHA (30 microM) decreased I(Ca(L)) by 48.9% and 45.2%, respectively. This inhibition was only partially reversible. In contrast, DIBHA (up to 50 microM) was devoided of effects on I(Ca(L)). 3. BHA inhibition of I(Ca(L)) was voltage-dependent and inversely related to the external concentration of Ca(2+). On the other hand, DTBHA inhibition was only voltage-dependent. 4. BHA and DTBHA shifted the voltage range of the steady-state inactivation curve to more negative potentials by 8 mV at the mid-potential of the curve, without affecting the activation curve. Furthermore, BHA and DTBHA did not modify the time-course of the current decay. 5. We conclude that the inhibition of I(Ca(L)) by BHA and DTBHA is qualitatively similar to that of a Ca(2+) channel blocker and is characterized by the stabilizing effect of the inactivated state of the channel.

2,5-Di-t-butyl-1,4-benzohydroquinone (BHQ) inhibits vascular L-type Ca(2+) channel via superoxide anion generation.

The aim of the present study was to investigate the effects of 2,5-di-t-butyl-1,4-benzohydroquinone (BHQ), an inhibitor of the sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA), on the whole-cell voltage-dependent L-type Ca(2+) current (I(Ca(L))) of freshly isolated smooth muscle cells from the rat tail artery using the patch-clamp technique. BHQ, added to the perfusion solution, reduced I(Ca(L)) in a concentration- (IC(50)=66.7 microM) and voltage-dependent manner. This inhibition was only partially reversible. BHQ shifted the voltage dependence of the steady-state inactivation curve to more negative potentials by 7 mV in the mid-potential of the curve, without affecting the activation curve as well as the time course of I(Ca(L)) inactivation. Preincubation of the cells either with 10 microM cyclopiazonic acid, a SERCA inhibitor, or with 3 mM diethyldithiocarbamate, an inhibitor of intracellular superoxide dismutase (SOD), did not modify BHQ inhibition of I(Ca(L)). On the contrary, this effect was no longer evident when SOD (250 u ml(-1)) was added to the perfusion medium. Either in the presence or in the absence of cells, BHQ gave rise to superoxide anion formation, which was markedly inhibited by the addition of SOD. These results indicate that, at micromolar concentrations, BHQ inhibits vascular I(Ca(L)) by giving rise to the formation of superoxide anion which in turn impairs the channel function.

Synthesis and antiperoxidant activity of new phenolic O-glycosides.

We describe the synthesis of some 3-tert-butyl-4-hydroxyphenyl D-glycopyranosides by reaction of tert-butylhydroquinone with beta-D-pentaacetyl-glucose, beta-D-pentaacetyl-galactose, 2-acetamido- and 3,4,6-tri-O-acetyl-2-butanamido-2-deoxy-beta-D-glucopyranosyl chlorides as well as the formation of anomeric 3-tert-butyl-4-hydroxyphenyl 4,6-di-O-acetyl-2,3-dideoxy-D-erythro-hex-2-eno-pyranosides by reaction between tert-butylhydroquinone and 3,4,6-tri-O-acetyl-D-glucal. All compounds, except 3-tert-butyl-4-hydroxyphenyl alpha- and beta-D-glucopyranosides, inhibited lipid peroxidation with a degree of potency comparable to that of tert-butyl hydroxyanisole.

Effects of taurine and some structurally related analogues on the central mechanism of thermoregulation: a structure-activity relationship study.

There is large body of evidences on the role of taurine in the central mechanisms of thermoregulation in mammals, but it is not clear, whether the hypothermic effect of taurine depends on its interaction with GABA receptors or with a specific receptor. In order to answer this question, we have performed a structure-activity relationship study by using both in vitro and in vivo preparations. MicroM amounts of taurine or each of 20 analogues were injected intracerebroventricularly in conscious, restrained rabbits while rectal temperature was recorded. Receptor-binding studies, with synaptic membrane preparations from rabbit brain were used to determine the affinities of these compounds for GABA(A) and GABA(B) receptors. Furthermore, the interaction with presynaptic GABA and taurine uptake systems was studied using crude synaptosomal preparations from rabbit brain. Among the compounds tested, (+/-)-cis-2-aminocyclohexanesulfonic acid, induced hypothermia, but did not interact with GABA(A) and GABA(B) receptors neither did it affect GABA and taurine uptake, thus suggesting that its effect on body temperature is not mediated by the central GABAergic system. Interestingly, the trans-isomer was devoid of effects either in vivo or in vitro. In order to explain (+/-)-cis-2-aminocyclohexanesulfonic acid-induced hypothermia, a stereoscopic model was produced showing its possible interactions with a putative taurine brain receptor.

L-type Ca(2+) channel current characteristics are preserved in rat tail artery myocytes after one-day storage.

AIM: To develop a cheap and simple method of storing for 24-h vascular tissue and single myocytes while preserving therein the biophysical and pharmacological characteristics of L-type Ca(2+) channels and contractile activity. METHODS: Rings or vascular smooth muscle cells obtained from the rat tail main artery were used either freshly (R0h and VSMC0h) or stored for 24 h (R24h and VSMC24h) at 4 °C, to record whole-cell L-type Ca(2+) currents (IC a(L) ) or measure contractile responses. RESULTS: R0h/VSMC0h and R24h/VSMC24h comparably contracted when stimulated with phenylephrine, high KCl or ATP. In both VSMC0h and VSMC24h, IC a(L) was identified and characterized as a stable inward current for at least 35 min; IC a(L) was comparably inhibited by the Ca(2+) antagonists nifedipine, verapamil and diltiazem and increased by the Ca(2+) channel agonist (S)-(-)-Bay K 8644; current density and current-voltage relationships were similar; at more hyperpolarized holding potentials, IC a(L) intensity increased comparably; nifedipine shifted the steady-state inactivation curve towards more negative potentials, while verapamil blocked IC a(L) in a frequency-dependent manner and slowed down the rate of recovery from inactivation in a comparable way. CONCLUSION: Findings show that smooth muscle contractile activity and the biophysical and pharmacological features of L-type Ca(2+) channels are similar in VSMC24h and VSMC0h. The fact that reproducible results were obtained in vascular myocytes up to 24 h after dissociation may facilitate vascular smooth muscle cell investigation by increasing throughput and reducing the number of animals required.

Quercetin relaxes rat tail main artery partly via a PKG-mediated stimulation of KCa 1.1 channels.

AIM: Protein kinases, activated by vasodilator substances, affect vascular function by regulating large conductance Ca(2+) -activated K(+) (KCa 1.1) channels. Thus, the aim of the present investigation was to address the hypothesis that quercetin-induced vasorelaxation is caused by a PKG-mediated stimulation of KCa 1.1 currents. METHODS: Single freshly isolated myocytes and endothelium-denuded rings of the rat tail main artery were employed for electrophysiological and contractility measurements respectively. RESULTS: Quercetin relaxed vessels and increased KCa 1.1 currents in a concentration-dependent manner: both effects were antagonized by the specific KCa 1.1 channel blocker iberiotoxin. Stimulation of KCa 1.1 currents was fully reversible upon drug washout, markedly reduced by Rp-8-Br-PET-cGMPs, a PKG-inhibitor, but not affected by catalase. Quercetin shifted by 34.3 mV the voltage dependence of KCa 1.1 channel activation towards more negative membrane potentials without affecting its slope. Under conditions of tight functional coupling between sarcoplasmic reticulum Ca(2+) release sites and KCa 1.1 channels, quercetin decreased both the frequency and the amplitude of KCa 1.1 transient currents in a ryanodine-like manner. CONCLUSION: The natural flavonoid quercetin relaxes the rat tail main artery partly via a PKG-mediated stimulation of smooth muscle KC a 1.1 channels.

The flavonoid scaffold as a template for the design of modulators of the vascular Ca(v) 1.2 channels.

BACKGROUND AND PURPOSE: Previous studies have pointed to the plant flavonoids myricetin and quercetin as two structurally related stimulators of vascular Ca(v) 1.2 channel current (I(Ca1.2) ). Here we have tested the proposition that the flavonoid structure confers the ability to modulate Ca(v) 1.2 channels. EXPERIMENTAL APPROACH: Twenty-four flavonoids were analysed for their effects on I(Ca1.2) in rat tail artery myocytes, using the whole-cell patch-clamp method. KEY RESULTS: Most of the flavonoids stimulated or inhibited I(Ca1.2) in a concentration- and voltage-dependent manner with EC(50) values ranging between 4.4 µM (kaempferol) and 16.0 µM (myricetin) for the stimulators and IC(50) values between 13.4 µM (galangin) and 100 µM [(±)-naringenin] for the inhibitors. Key structural requirements for I(Ca1.2) stimulatory activity were the double bond between C2 and C3 and the hydroxylation pattern on the flavonoid scaffold, the latter also determining the molecular charge, as shown by molecular modelling techniques. Absence of OH groups in the B ring was key in I(Ca1.2) inhibition. The functional interaction between quercetin and either the stimulator myricetin or the antagonists resokaempferol, crysin, genistein, and 5,7,2'-trihydroxyflavone revealed that quercetin expressed the highest apparent affinity, in the low µM range, for Ca(v) 1.2 channels. Neither protein tyrosine kinase nor protein kinase Cα were involved in quercetin-induced stimulation of I(Ca1.2). CONCLUSIONS AND IMPLICATIONS: Quercetin-like plant flavonoids were active on vascular Ca(v)1.2 channels. Thus, the flavonoid scaffold may be a template for the design of novel modulators of vascular smooth muscle Ca(v)1.2 channels, valuable for the treatment of hypertension and stroke.

GABA-mediated effects of some taurine derivatives injected i.c.v. on rabbit rectal temperature and gross motor behavior.

Some synthetic taurine analogues, namely ethanolamine-O-sulphate (EOS), N,N-dimethyltaurine (DMT), N,N,N-trimethyltaurine (TMT) and 2-aminoethylphosphonic acid (AEP) were shown to interact with rabbit brain GABA(A)- or GABA(B)-receptors, while (+/-)piperidine-3-sulfonic acid (PSA) inhibited the activity of rabbit brain 4-aminobutyrate transaminase. This suggests that they behave like direct/indirect GABA agonists or GABA antagonists and affect thermoregulation and gross motor behaviour (GMB) which are under GABA control. In the present study micromole (1.2-48) amounts of these compounds were i.c.v. injected in conscious, restrained rabbits while monitoring rectal temperature (RT), ear skin temperature (EST) and GMB. AEP, EOS, DMT and TMT induced a dose-related hyperthermia, ear vasoconstriction and excitation of GMB, while PSA induced a dose-related hypothermia, ear vasodilation and inhibition of GMB. EOS antagonized in a dose-related fashion hypothermia induced by 60 nmol THIP, a GABA(A) agonist, while AEP, DMT and TMT counteracted that induced by 8 nmol R(-)Baclofen, a GABA(B) agonist. In conclusion, EOS and AEP, DMT, TMT seem to act as GABA(A) and GABA(B) antagonists, respectively, while PSA behaves like an indirect GABA agonist, all affecting the central mechanisms which drive rabbit thermoregulation.

Characterization of voltage-gated calcium currents in freshly isolated smooth muscle cells from rat tail main artery.

The aim of the present study was to characterize voltage-gated Ca2+ currents in smooth muscle cells freshly isolated from rat tail main artery in the presence of 5 mmol L(-1) external Ca2+. Calcium currents were identified on the basis of their voltage dependencies and sensitivity to nifedipine, Ni2+ and cinnarizine. In the majority of the cells studied, T- and L-type currents were observed, while the remaining cells showed predominantly L-type currents. In the latter group of cells, holding potential change from -50 to either -70 or -90 mV increased the corresponding inward current amplitude while its voltage activation threshold remained unchanged. The steady state inactivation of L-type Ca2+ channels showed half-maximal inactivation at -38 mV. A Ca2+-dependent inactivation was also evident. Nifedipine (3 micromol L(-1)) blocked L-type but not T-type Ca2+ currents. Ni2+ (50 micromol L(-1)) as well as cinnarizine (1 micromol L(-1)) suppressed the nifedipine-resistant, T-type component of the currents. At higher concentrations, both Ni2+ (0.3-1 mmol L(-1)) and cinnarizine (10 micromol L(-1)) blocked the net inward current. Replacement of Ca2+ with 10 mmol L(-)1 Ba2+ significantly increased the amplitude of L-type Ca2+ currents. These results demonstrate that smooth muscle cells freshly isolated from rat tail main artery may be divided into two populations, one expressing both L- and T-type and the other only L-type Ca2+ channels. Furthermore, this report shows that in arterial smooth muscle cells cinnarizine potently inhibited T-type currents at low concentrations (1 micromol L(-1)) but also blocked L-type Ca2+ currents at higher concentrations (10 micromol L(-1)).