1,721,036 research outputs found
Quercetin antagonism of Bay K 8644 effects on rat tail artery L-type Ca(2+) channels
No full textThe functional interaction between two L-type Ca(2+) channel activators, quercetin and (S)-(-)-methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)pyridine-5-carboxylate (Bay K 8644), has been investigated in vascular smooth muscle cells. L-type Ca(2+) currents [I(Ca(L))] were recorded in freshly isolated rat tail main artery myocytes using the whole-cell patch-clamp method. Bay K 8644 increased I(Ca(L)) in a concentration-dependent manner with a pEC(50) value of 8.25. Pre-incubation of myocytes with concentrations of quercetin per se ineffective as an L-type Ca(2+) channel activator (0.1 and 0.3 microM) inhibited significantly the maximal response evoked by Bay K 8644, but left unaltered its potency. Quercetin (0.1 microM) prevented the hyperpolarizing shift of the steady-state inactivation curve induced by 0.1 microM Bay K 8644 and its stimulation of I(Ca(L)) tail current intensity without modifying Bay K 8644-induced effects on I(Ca(L)) activation, inactivation, deactivation kinetics as well as on use-dependence and recovery from inactivation. Quercetin at nutritionally meaningful concentrations, limited the responsiveness of vascular L-type Ca(2+) channels to the pharmacological stimulation operated by Bay K 8644. These data contribute to a better understanding of quercetin effects on experimental in vivo cardioprotection
The vasodilator papaverine stimulates L-type Ca(2+) current in rat tail artery myocytes via a PKA-dependent mechanism
No full textPapaverine is an opium alkaloid, primarily used as an antispasmodic drug and as a cerebral and coronary vasodilator. Its phosphodiesterase inhibitory activity promotes increase of cAMP levels mainly in the cytosol. As cAMP is known to modulate L-type Ca(2+) channel activity, here we tested the proposition that papaverine could affect vascular channel function. An in-depth analysis of the effect of papaverine on Ba(2+) or Ca(2+) current through L-type Ca(2+) channel [IBa(L) or ICa(L)], performed in rat tail artery myocytes using either the whole-cell or the perforated patch-clamp method, was accompanied by a functional study on rat aorta rings. Papaverine increased current amplitude under both the perforated or whole-cell configuration. Stimulation of the current by papaverine was concentration-, Vh-, frequency-, and charge carrier-dependent, and fully reverted by drug washout. The PKA inhibitor H89, but not the PKG inhibitor Rp-8-Br-cGMPS, antagonised papaverine- as well as IBMX- (another phosphodiesterase inhibitor) induced IBa(L) stimulation. In cells pre-treated with IBMX, application of papaverine failed to increase current amplitude. Papaverine sped up the inactivation kinetics of IBa(L), though only at concentrations ≥30μM, and shifted the voltage dependence of the inactivation curve to more negative potentials. In rings, the vasorelaxing activity of papaverine was enhanced by previous treatment with nifedipine. In conclusion, papaverine stimulates vascular L-type Ca(2+) channel via a PKA-dependent mechanism, thus antagonising its main vasodilating activity
Human Tuberculosis. III. Current and Prospective Approaches in Anti-Tubercular Therapy
No full textIneffectively treated tuberculosis (TB) is associated with substantial morbidity and mortality. Cure of TB patients is hampered by the development of multidrug resistance in M. tuberculosis and the need of long-term treatment. The diarylquinoline derivative bedaquiline was approved in December 2012 under the accelerated-approval regulations of FDA as part of a combination therapy for treating adults with pulmonary MDR-TB for whom effective cures are not otherwise available. The bicyclic nitroimidazoles delamanid and its companion pretomanid inhibit mycolic acid synthesis via an unknown mechanism. In November 2013, delamanid received conditional approval by the European Medicines Agency for MDR-TB treatment. Use of both drugs, however, is limited owing to toxicity issues. If the aim to reduce treatment duration is pursued in order to limit costs and improve patient adherence, it is mandatory to demonstrate their noninferiority with fewer months of therapy. In three phase III clinical trials the efficacy of the most recent fluoroquinolones, gatifloxacin and moxifloxacin, has been investigated in a four-month treatment regimen of drug-susceptible TB. In all three studies, after two months the culture conversion rates of observed sputum indicated that fluoroquinolone-based therapies were likely to be superior. However, this feature did not reliably predict sterilizing activity or a risk of relapse. In other words, the shortened treatments were not noninferior to standard treatments. To counteract mycobacterial survival strategies and reduce the timelength of treatment with anti-TB drugs, other novel and powerful agents, as well as tuberculosis vaccines, are under intense clinical investigation for safety and efficacy assessment
Effects of quercetin and rutin on vascular preparations: a comparison between mechanical and electrophysiological phenomena.
No full textBackground: Several studies have indicated that quercetin promotes relaxation of vascular smooth muscle both in vivo and in vitro. However, Saponara et al. [(2002) Br J Pharmacol 135:1819-1827] have demonstrated that quercetin is an activator of vascular L-type Ca2+ channels. Aim of the study: We investigated the mechanical and electrophysiological properties of quercetin and its rutoside, rutin, in an attempt to clarify how Ca2+ channel activation might be related to the myorelaxing activity. Methods: Aorta ring preparations and single tail artery myocytes were employed for functional and patch-clamp experiments, respectively. Results: Rutin was found to relax intact rat aorta rings, which had been precontracted with phenylephrine (pIC50 = 5.65±0.3 1) but in contrast had no effect on depolarised (60 mM K+) preparations or on those from which the endothelium had been removed. Furthermore, rutin did not affect L-type Ca2+ current recorded in rat tail artery myocytes. The quercetin-induced relaxation of intact rings precontracted with phenylephrine exhibited two components characterised by 6.23±0.38 and 4.66±0.09 pIC50, respectively. Removal of the endothelium abolished the first component, leaving the second unaltered. Moreover, quercetin was found to relax 60 mM K+ depolarised rings with a pIC50 of 4.59±0.03. The application of quercetin in isolated smooth muscle cells brought about a marked increase of L-type Ca2+ current (pEC50= 5.09±0.05). Unlike quercetin, Bay K 8644 contracted aorta rings preincubated with 10, 20 or 30 mM K+. The myotonic effect of Bay K 8644 was observed both in the absence or presence of 30 μM quercetin. The application of Bay K 8644 (10-100 nM) caused a further significant increase in L-type Ca2+ current in rat tail artery myocytes stimulated with 30 μM quercetin. Conclusions: Quercetin is a naturally occurring L-type Ca2+ channel agonist. This effect, however, is overwhelmed by quercetin-induced vasorelaxation taking place via pathways which are more relevant than L-type Ca2+ influx in the hierarchy of functional competencies
In vitro assessement of tariquidar toxicity towards vasculature
No full textBackground: The P-glycoprotein (P-gp) inhibitor tariquidar, intravenously administered, increases brain uptake of radiolabeled P-gp
substrates, commonly used to detect functional alterations of blood –
brain barrier pumps in PET imaging [1]. However, the doses that are
required — up to 4-fold higher than those already used in clinical trials
to reverse multidrug resistance — cause syncopal episodes and hypotension [2]. Therefore, we investigated the toxic hazard of these
doses towards the vasculature.
Methods: The effects of tariquidar on A7r5 and EA.hy926 cell
viability, on the mechanical activity of fresh and cultured rat aorta
rings, as well as on A7r5 CaV1.2 channel current (ICa1.2) were
analysed [3].
Results: In both A7r5 and EA.hy926 cells, tariquidar was generally
devoid of cytotoxic effects up to a concentration of 1 μM. However, at
10 μM, it caused apoptosis already after 24 h treatment. In endothelium-denuded aorta rings, 10 μM tariquidar relaxed contractions
induced by phenylephrine but not by high K+. The contractile activity
of rings cultured for 7 days was not affected by drug treatment.
Finally, tariquidar did not modify ICa1.2 intensity and kinetics.
Discussion: Tariquidar exerts both cytotoxic and acute vascular
effects at concentrations comparable to those employed in PET
imaging. This may limit its use as diagnostic tool.
Acknowledgements: This work was supported by the Italian Ministry
for Instruction, Universities and Research (Futuro in Ricerca 2012,
RBFR12SOQ1 to S. S.)
The surge of flavonoids as novel, fine regulators of cardiovascular Cav channels
Full text linkIon channels underlie a wide variety of physiological processes that involve rapid changes in cell dynamics, such as cardiac and vascular smooth muscle contraction. Overexpression or dysfunction of these membrane proteins are the basis of many cardiovascular diseases that represent the leading cause of morbidity and mortality for human beings. In the last few years, flavonoids, widely distributed in the plant kingdom, have attracted the interest of many laboratories as an emerging class of fine ion, in particular Cav, channels modulators. Pieces of in vitro evidence for direct as well as indirect effects exerted by various flavonoids on ion channel currents are now accumulating in the scientific literature. This activity may be responsible, at least in part, for the beneficial and protective effects of dietary flavonoids toward cardiovascular diseases highlighted in several epidemiological studies. Here we examine numerous studies aimed at analysing this feature of flavonoids, focusing on the mechanisms that promote their sometimes controversial activities at cardiovascular Cav channels. New methodological approaches, such as molecular modelling and docking to Cav1.2 channel α1c subunit, used to elucidate flavonoids intrinsic mechanism of action, are introduced. Moreover, flavonoid-membrane interaction, bioavailability, and antioxidant activity are taken into account and discussed
Mechanisms of the antispasmodic activity of 3,5-di-t-butyl catechol (DTCAT) on rat vascular smooth muscles
No full textIn skeletal muscle sarcoplasmic reticulum vesicles, 3,5-di-t-butyl catechol (DTCAT) promotes the release of Ca(2+) through the activation of ryanodine receptor Ca(2+) channels. DTCAT mechanical and electrophysiological effects have now been investigated in rat aorta rings and single tail artery myocytes. Rat aorta rings incubated with either 30 microM ryanodine or 100 microM DTCAT developed tension, which averaged 36% and 7%, respectively, of that induced by phenylephrine. DTCAT reduced concentration-dependently both aorta ring contractions to high K(+) (IC(50)=13.5 microM) and L-type Ba(2+) current (IC(50)=22.0 microM) in isolated myocytes. Tetraethylammonium did not reverse the antispasmodic effect of DTCAT in rings stimulated with either 25 or 60 mM K(+). DTCAT relaxed concentration-dependently phenylephrine-pre-contracted rings with intact endothelium (IC(50)=10.9 microM). This effect was markedly reduced by pre-incubation of rings with 100 microM Nomega-nitro-l-arginine methyl ester. DTCAT antagonised phenylephrine-induced contractions in endothelium-deprived rings, either in the presence or in the absence of ryanodine (IC(50)=18.7 microM and 39.8 microM, respectively). Furthermore, both DTCAT (IC(50)=53.3 microM) and ryanodine reduced significantly the response to phenylephrine in the absence of extracellular Ca(2+). Phenylephrine-stimulated influx of extracellular Ca(2+) was markedly inhibited when tissues were pre-treated with DTCAT (IC(50)=19.0 microM) as well as nifedipine. DTCAT (>100 microM) was also able to antagonise the contractions induced by phorbol 12-myristate, 13-acetate. In conclusion, this is the first demonstration that DTCAT inhibits vascular smooth muscle voltage-operated Ca(2+) channels and promotes the release of endothelial nitric oxide. Ryanodine receptor Ca(2+) channels activation or the impairment of the contractile apparatus by DTCAT seem to play a secondary role in its vascular activity
Quercetin as a novel activator of L-type Ca2+ channels in rat tail artery smooth muscle cells
No full text1. The aim of this study was to investigate the effects of quercetin, a natural polyphenolic flavonoid, on voltage-dependent Ca(2+) channels of smooth muscle cells freshly isolated from the rat tail artery, using either the conventional or the amphotericin B-perforated whole-cell patch-clamp method. 2. Quercetin increased L-type Ca(2+) current [I(Ca(L))] in a concentration- (pEC(50)=5.09+/-0.05) and voltage-dependent manner and shifted the maximum of the current-voltage relationship by 10 mV in the hyperpolarizing direction, without, however, modifying the threshold and the equilibrium potential for Ca(2+). 3. Quercetin-induced I(Ca(L)) stimulation was reversible upon wash-out. T-type Ca(2+) current was not affected by quercetin. Quercetin shifted the voltage dependence of the steady-state inactivation and activation curves to more negative potentials by about 5.5 and 7.5 mV respectively, in the mid-potential of the curves as well as increasing the slope of activation. Quercetin slowed both the activation and the deactivation kinetics of the I(Ca(L)). The inactivation time course was also slowed but only at voltages higher than 10 mV. Moreover quercetin slowed the rate of recovery from inactivation. 4. These results prove quercetin to be a naturally-occurring L-type Ca(2+) channel activator
Failure Mode and Effect Analysis of Patch-clamp Laboratory Instrumentation for Electrophysiology Measurements
No full textThe patch-clamp technique is an electrophysiological method that allows the recording of the macroscopic whole-cell current as well as of the microscopic single-channel current, flowing across biological membranes through ion channels. Though different experimental preparations exist, and various types of experiments can be carried out with this technique, some basic instrumentation is common to all patch-clamp workstations: the vibration isolation table, the Faraday cage, the microscope, the micromanipulator, the amplifier, the pc, the pipette puller, and the microforge. Failures in the electronic instrumentation can markedly affect current recordings, as their amplitudes usually range between a few pA and a few nA, thus influencing the accurate assessment of the effect of drugs on ion channels. In this paper different types of failure that may occur in the electronic instrumentation for the patch-clamp are discussed. Moreover, an analysis of their effect on the acquired measurements during electrophysiology experiments is carried ou
2,5-Di-t-butyl-1,4-benzohydroquinone (BHQ) inhibits vascular L-type Ca2+ channel via superoxide anion generation
No full textThe 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
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