Giampietro Sgaragli

Quercetin as a novel activator of L‐type Ca2+ channels in rat tail artery smooth muscle cells

The aim of this study was to investigate the effects of quercetin, a natural polyphenolic flavonoid, on voltage‐dependent Ca2+ 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. Quercetin increased L‐type Ca2+ current [ICa(L)] in a concentration‐ (pEC50=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 Ca2+. Quercetin‐induced ICa(L) stimulation was reversible upon wash‐out. T‐type Ca2+ 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 ICa(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. These results prove quercetin to be a naturally‐occurring L‐type Ca2+ channel activator.

Red wine alcohol promotes quercetin absorption and directs its metabolism towards isorhamnetin and tamarixetin in rat intestine in vitro.

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. 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 37°C, after which the mucosa was removed and processed for HPLC analysis. Tissues exposed to red wine had significantly higher amounts of both quercetin (× −3; P<0.001) and quercetin‐3‐O‐glucoside (× −1.5; P<0.01) 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 (P<0.05; P<0.01, respectively). 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 (P<0.01 and P<0.001, respectively). 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.

Interactions of taurine and structurally related analogues with the GABAergic system and taurine binding sites of rabbit brain

The aim of this study was to find taurinergic compounds that do not interact with brain GABA ergic systems. Washed synaptic membranes (SM) from whole rabbit brain were able to bind [3H]muscimol. Saturation experiments of the binding of [3H]GABA to GABAB receptors showed that SM possess two binding components; twice Triton X‐100‐treated SM contained 0.048 mmol endogenous taurine/kg protein and bound [3H]taurine in a saturable manner (Kd=249.0±6.3 nM and Bmax=3.4±1.0 pmol mg−1 prot). Among the 19 structural analogues of taurine, 6‐aminomethyl‐3‐methyl‐4H‐1,2,4‐benzothiadiazine 1,1‐dioxide (TAG), 2‐aminoethylarsonic (AEA), 2‐hydroxyethanesulfonic (ISE) and (±)cis‐2‐aminocyclohexane sulfonic acids (CAHS) displaced [3H]taurine binding (Ki=0.13, 0.13, 13.5 and 4.0 μM, respectively). These analogues did not interact with GABAA and GABAB receptors and did not affect taurine‐ and GABA‐uptake systems and GABA‐transaminase activity. 3‐Aminopropanesulfonic acid (OMO), β‐alanine, pyridine‐3‐sulfonic acid, N,N,N‐trimethyltaurine (TMT), 2‐(guanidino)ethanesulfonic acid (GES), ethanolamine‐O‐sulphate, N,N‐dimethyltaurine (DMT), taurine and (±)piperidine‐3‐sulfonic acid (PSA) inhibited [3H]muscimol binding to GABAA receptors with different affinities (Ki=0.013, 7.9, 24.6, 47.5, 52.0, 91.0, 47.5, 118.1 and 166.3 μM, respectively). Taurine, 2‐aminoethylphosphonic acid, DMT, TMT and OMO inhibited the binding of [3H]GABA to GABAB receptors with Kis in the μM range (0.8, 3.5, 4.4, 11.3 and 5.0, respectively). GES inhibited taurine uptake (IC50=3.72 μM) and PSA GABA transaminase activity (IC50=103.0 μM). In conclusion, AEA, TAG, ISE and CAHS fulfill the criteria for taurinergic agents.

2,5-Di-t-butyl-1,4-benzohydroquinone (BHQ) inhibits vascular L-type Ca2+ 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 Ca2+‐ATPase (SERCA), on the whole‐cell voltage‐dependent L‐type Ca2+ current (ICa(L)) of freshly isolated smooth muscle cells from the rat tail artery using the patch‐clamp technique. BHQ, added to the perfusion solution, reduced ICa(L) in a concentration‐ (IC50=66.7 μM) 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 ICa(L) inactivation. Preincubation of the cells either with 10 μM cyclopiazonic acid, a SERCA inhibitor, or with 3 mM diethyldithiocarbamate, an inhibitor of intracellular superoxide dismutase (SOD), did not modify BHQ inhibition of ICa(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 ICa(L) by giving rise to the formation of superoxide anion which in turn impairs the channel function.

A specific taurine recognition site in the rabbit brain is responsible for taurine effects on thermoregulation

Taurine and GABA are recognized as endogenous cryogens. In a previous study, some structural analogues of taurine, namely 6‐aminomethyl‐3‐methyl‐4H‐1,2,4‐benzothiadiazine 1,1‐dioxide (TAG), 2‐aminoethylarsonic (AEA), 2‐hydroxyethanesulfonic (ISE) and (±)cis‐2‐aminocyclohexane sulfonic acids (CAHS) have been shown to displace [3H]taurine binding from rabbit brain synaptic membrane preparations, without interacting either with GABA‐ergic systems, nor with taurine uptake mechanism, thus behaving like direct taurinergic agents. To answer the question whether the role of taurine as an endogenous cryogen depends on the activation of GABA receptors or that of specific taurine receptor(s), taurine or the above structural analogues were injected intracerebroventricularly in conscious, restrained rabbits singularly or in combination and their effects on rectal (RT)‐ and ear–skin temperature and gross motor behavior (GMB) were monitored. Taurine (1.2 × 10−6–4.8 × 10−5 mol) induced a dose‐related hypothermia, vasodilation at ear vascular bed and inhibition of GMB. CAHS, at the highest dose tested (4.8 × 10−5 mol) induced a taurine‐like effect either on RT or GMB. On the contrary ISE, injected at the same doses of taurine, induced a dose‐related hyperthermia, vasoconstriction and excitation of GMB. AEA and TAG caused a dose‐related hyperthermia, but at doses higher than 1.2 × 10−7 mol caused death within 24 h after treatment. CAHS (4.8 × 10−5 mol) antagonized the hyperthermic effect induced by TAG (1.2 × 10−6 mol), AEA (1.2 × 10−8 mol) or ISE (4.8 × 10−5 mol). In conclusion, these findings may indicate the existence of a recognition site specific for taurine, responsible for its effects on thermoregulation.

3,5‐Dibenzoyl‐4‐(3‐phenoxyphenyl)‐1,4‐dihydro‐2,6‐dimethylpyridine (DP7) as a new multidrug resistance reverting agent devoid of effects on vascular smooth muscle contractility

The aim of this study was to investigate the effects of 3,5‐diacetyl‐ (DP1–DP5) and 3,5‐dibenzoyl‐1,4‐dihydropyridines (DP6–DP11) on vascular functions in vitro, by comparing their mechanical and electrophysiological actions in rat aorta rings and single rat tail artery myocytes, respectively, and to quantify their multidrug resistance (MDR)‐reversing activity in L5178 Y mouse T‐lymphoma cells transfected with MDR1 gene. In rat aorta, the 11 compounds tested, but 3,5‐dibenzoyl‐4‐(3‐phenoxyphenyl)‐1,4‐dihydro‐2,6‐dimethylpyridine (DP7), 3,5‐dibenzoyl‐4‐(3‐chlorophenyl)‐1,4‐dihydro‐2,6‐dimethylpyridine (DP9), 3,5‐dibenzoyl‐4‐(4‐chlorophenyl)‐1,4‐dihydro‐2,6‐dimethylpyridine (DP10) and 3,5‐dibenzoyl‐4‐phenyl‐1,4‐dihydro‐2,6‐dimethylpyridine (DP11), antagonized 60 mM K+ (K60)‐induced contraction in a concentration‐dependent manner, with IC50 (M) values ranging between 5.65 × 10−7 and 2.23 × 10−5. The 11 dihydropyridines tested, but DP7, inhibited L‐type Ca2+ current recorded in artery myocytes in a concentration‐dependent manner, with IC50 (M) values ranging between 1.12 × 10−6 and 6.90 × 10−5. The K+‐channel opener cromakalim inhibited the Ca2+‐induced contraction in K30 but not that evoked in K60. On the contrary, DP7 was ineffective in both experimental conditions. When the rings were preincubated with 1 mM Ni2+ plus 1 μM nifedipine, the response to phenylephrine was significantly reduced by 2,5‐di‐t‐butyl‐1,4‐benzohydroquinone (BHQ), a well‐known endoplasmic reticulum Ca2+‐ATPase inhibitor. DP7 had no effects on this model system. In L5178 MDR cell line, the 11 dihydropyridines tested, but 3,5‐diacetyl‐4‐(2‐nitrophenyl)‐1,4‐dihydro‐2,6‐dimethylpyridine (DP1), 3,5‐diacetyl‐4‐(3‐phenoxyphenyl)‐1,4‐dihydro‐2,6‐dimethylpyridine (DP2) and 3,5‐diacetyl‐4‐(3‐chlorophenyl)‐1,4‐dihydro‐2,6‐dimethylpyridine (DP4), exhibited an MDR‐reversing activity, with IC50 values ranging between 3.02 × 10−7 and 4.27 × 10−5, DP7 being the most potent. In conclusion, DP7 may represent a lead compound for the development of potent dihydropyridine MDR chemosensitizers devoid of vascular effects.

N,N-bis(cyclohexanol)amine aryl esters: a new class of highly potent transporter-dependent multidrug resistance inhibitors.

A new series of Pgp-dependent MDR inhibitors having a N,N-bis(cyclohexanol)amine scaffold was designed on the basis of the frozen analogue approach. The scaffold chosen gives origin to different geometrical isomers. The new compounds showed a wide range of potencies and efficacies on doxorubicin-resistant erythroleukemia K562 cells in the pirarubicin uptake assay. The most interesting compounds (isomers of 3) were studied further evaluating their action on the ATPase activity present in rat small intestine membrane vesicles and doxorubicin cytotoxicity potentiation on K562 cells. The latter assay was performed also on the isomers of 4. The four isomers of each set present different behavior in each of these tests. Compound 3d shows the most promising properties as it was able to completely reverse Pgp-dependent pirarubicin extrusion at low nanomolar concentration, inhibited ATPase activity at 5 x 10(-9) and increased the cytotoxicity of doxorubicin with a reversal fold (RF) of 36.4 at 3 microM concentration.

Mechanism of osthole inhibition of vascular Cav1.2 current

Osthole is a coumarin extracted from Cnidium monnieri (L.) Cusson. The medicinal plant is widely used in Vietnamese as well as Chinese traditional medicine as a vasodilating and antihypertensive agent. Here we have tested the proposition that the block of Ca(v)1.2 channels is mainly responsible for its vascular activity. An in-depth analysis of the effect of osthole on Ca(v)1.2 current (I(Ca1.2)) was performed in rat tail artery myocytes using the whole-cell patch-clamp method. Osthole decreased I(Ca1.2) in a concentration- and voltage-dependent manner. At holding potentials of -50 and -80mV, the pIC(50) values were 4.78±0.07 and 4.36±0.08, respectively; the latter corresponded to the drug apparent dissociation constant for resting channels, K(R), of 47.8μM. Osthole speeded up the inactivation kinetics of I(Ca1.2) and shifted the voltage dependence of the inactivation curve to more negative potentials in a concentration-dependent manner, with an apparent dissociation constant for inactivated channels (K(I)) of 6.88μM. Block of I(Ca1.2) was frequency-dependent and the rate of recovery from inactivation was slowed down. In conclusion, osthole is a vascular Ca(v)1.2 channel antagonist stabilizing the channel in its inactivated state. This mechanism may account for the systolic blood pressure reduction induced by the drug in animal models of hypertension and points to osthole as a lead for the development of novel antihypertensive agents.

Changes in rectal temperature and ECoG spectral power of sensorimotor cortex elicited in conscious rabbits by i.c.v. injection of GABA, GABAA and GABAB agonists and antagonists

In order to ascertain whether both GABAA and GABAB, or only GABAB receptors, directly modulate thermoregulation in conscious rabbits, GABAA/GABAB agonist and antagonist agents were injected intracerebroventricularly in conscious rabbits while monitoring changes in rectal temperature (RT), gross motor behaviour (GMB) and electrocorticogram (ECoG) power spectra (ps) from sensorimotor cortices. GABA (48 μmol), nipecotic acid (50 nmol), THIP (60 nmol), muscimol (18 nmol) and baclofen (8 nmol) induced hypothermia (−ΔRTmax values of 1.70±0.1, 1.4±0.2, 1.0±0.4, 1.1±0.2 and 1.6±0.3°C, respectively), accompanied by inhibition of GMB and ECoG synchronization. THIP increased ps at δ frequency band (1.1−3.3 Hz), while GABA, nipecotic acid, muscimol and baclofen did the same at both δ and θ (4.6−6.5 Hz) frequency bands. ECoG ps changes were concomitant or even preceded hypothermia. Bicuculline (1.8 nmol) induced hyperthermia (ΔRTmax 1.2±0.5°C) and slight excitation of GMB, while CGP35348 (1.2 μmol) did not affect RT nor GMB. Both compounds did not affect ECoG ps. Bicuculline potentiated muscimol‐induced hypothermia, inhibition of GMB and synchronization of ECoG, while CGP35348 fully antagonized these effects. In conclusion, the present results, while confirming the prevailing role of GABAB, also outline a direct involvement of GABAA receptors in the central mechanisms of thermoregulation. Ascending inhibition towards discrete cortical areas controlling muscular activity and thermogenesis may result from GABA receptor activation in neurones proximal to the ventricles, thus contributing to hypothermia, although hypothermia‐induced reduction of neuronal activity of these cortical areas cannot be ruled out.

Homotaurine and muscimol mimic taurine and GABA effects on muscle tone and temperature regulation.

SummaryTaurine, homotaurine, GABA and muscimol, given intraventricularly to the conscious, unrestrained rabbit cause hypothermia and a reduction in skeletal muscle tone. Taurine and homotaurine desynchronize areas of the motor and limbic cortices, while GABA and muscimol synchronize both tracings and markedly depress the arousal reaction following external stimuli.