In our experimental model, bone marrow-derived mesenchymal stem cells injection improved muscle regeneration and increased contractile function of anal sphincters after injury and repair. Therefore, mesenchymal stem cells may represent an attractive tool for treating anal sphincter lesions in humans. Investigations into the biologic basis of this phenomenon should increase our knowledge on underlying mechanisms involved in sphincter repair.

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 (pIC50 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‐β‐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 Ca2+‐activated K+ (BKCa) currents in a concentration‐dependent manner; this stimulation was iberiotoxin‐sensitive and fully reversible upon drug wash‐out. (+/−)‐Naringenin accelerated the activation kinetics of BKCa 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 BKCa current was insensitive either to changes in the intracellular Ca2+ concentration or to the presence, in the pipette solution, of the fast Ca2+ 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 BKCa channels in myocytes. British Journal of Pharmacology (2006) 149, 1013–1021. doi:

1 The aim of this study was to investigate the e ects of quercetin, a natural polyphenolic¯avonoid, 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+ . Quercetin-induced I Ca(L) stimulation was reversible upon wash-out. T-type Ca 2+ current was not a ected by quercetin. 3 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.

BACKGROUND AND PURPOSEPrevious studies have pointed to the plant flavonoids myricetin and quercetin as two structurally related stimulators of vascular Cav1.2 channel current (ICa1.2). Here we have tested the proposition that the flavonoid structure confers the ability to modulate Cav1.2 channels. EXPERIMENTAL APPROACHTwenty-four flavonoids were analysed for their effects on ICa1.2 in rat tail artery myocytes, using the whole-cell patch-clamp method. KEY RESULTSMost of the flavonoids stimulated or inhibited ICa1.2 in a concentration-and voltage-dependent manner with EC50 values ranging between 4.4 mM (kaempferol) and 16.0 mM (myricetin) for the stimulators and IC50 values between 13.4 mM (galangin) and 100 mM [(Ϯ)-naringenin] for the inhibitors. Key structural requirements for ICa1.2 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 ICa1.2 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 mM range, for Cav1.2 channels. Neither protein tyrosine kinase nor protein kinase Ca were involved in quercetin-induced stimulation of ICa1.2. CONCLUSIONS AND IMPLICATIONSQuercetin-like plant flavonoids were active on vascular Cav1.2 channels. Thus, the flavonoid scaffold may be a template for the design of novel modulators of vascular smooth muscle Cav1.2 channels, valuable for the treatment of hypertension and stroke. AbbreviationsBay K 8644, (S)-(-)-methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)pyridine-5-carboxylate; ICa1.2, Cav1.2 channel current; Vh, holding potential

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1 Moderate consumption of red wine has been associated with beneficial effects on human health, and this has been attributed to the flavonoid content. Factors that influence the bioavailability of this group of polyphenolic compounds are therefore important. 2 Using the rat cannulated everted jejunal sac technique, we have investigated the effect of alcohol on the intestinal absorption of quercetin and its 3-O-glucoside from red wine. Tissue preparations were incubated in whole or dealcoholised red wine, diluted 1 : 1 with Krebs buffer for 20 min at 371C, after which the mucosa was removed and processed for HPLC analysis. Tissues exposed to red wine had significantly higher amounts of both quercetin ( Â 3; Po0.001) and quercetin-3-O-glucoside ( Â 1.5; Po0.01) associated with them, compared with sacs incubated in the dealcoholised equivalent. In addition, both tamarixetin (T) and isorhamnetin (I), in the mucosal tissue from sacs exposed to the whole wine, were significantly elevated approximately two fold (Po0.05; Po0.01, respectively). 3 Similar results were obtained when sacs were incubated in Krebs buffer containing a mixture of pure quercetin and quercetin-3-O-glucoside with or without alcohol, and, although effects on the apparent absorption of Q and Q-3-G were not so marked, concentrations of the metabolites quercetin-3-O-glucuronide and I were significantly increased by the presence of alcohol (Po0.01 and Po0.001, respectively). 4 It is therefore plausible that the moderate alcohol content of red wine contributes to its beneficial health effects in humans by both increasing the absorption of quercetin and quercetin-3-O-glucoside and by channelling their metabolism towards O-methylation to yield compounds (T and I), which have potential protective effects against cancer and cardiovascular diseases.

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