Simona Saponara

(+/−)-Naringenin as large conductance Ca2+-activated K+ (BKCa) channel opener in vascular smooth muscle cells

The aim of this study was to investigate, in vascular smooth muscle cells, the mechanical and electrophysiological effects of (+/−)‐naringenin.

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.

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.

Targeting Dopamine D3 and Serotonin 5-HT1A and 5-HT2A Receptors for Developing Effective Antipsychotics: Synthesis, Biological Characterization, and Behavioral Studies

Combination of dopamine D3 antagonism, serotonin 5-HT1A partial agonism, and antagonism at 5-HT2A leads to a novel approach to potent atypical antipsychotics. Exploitation of the original structure-activity relationships resulted in the identification of safe and effective antipsychotics devoid of extrapyramidal symptoms liability, sedation, and catalepsy. The potential atypical antipsychotic 5bb was selected for further pharmacological investigation. The distribution of c-fos positive cells in the ventral striatum confirmed the atypical antipsychotic profile of 5bb in agreement with behavioral rodent studies. 5bb administered orally demonstrated a biphasic effect on the MK801-induced hyperactivity at dose levels not able to induce sedation, catalepsy, or learning impairment in passive avoidance. In microdialysis studies, 5bb increased the dopamine efflux in the medial prefrontal cortex. Thus, 5bb represents a valuable lead for the development of atypical antipsychotics endowed with a unique pharmacological profile for addressing negative symptoms and cognitive deficits in schizophrenia.

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.

The flavonoid scaffold as a template for the design of modulators of the vascular Cav1.2 channels

BACKGROUND AND PURPOSE Previous 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.

Quercetin antagonism of Bay K 8644 effects on rat tail artery L-type Ca2+ channels

The 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.

Cardamonin Is a Bifunctional Vasodilator that Inhibits Cav1.2 Current and Stimulates KCa1.1 Current in Rat Tail Artery Myocytes

An in-depth analysis of the effects of cardamonin, 2′,4′-dihydroxy-6′-methoxychalcone, on rat tail artery preparations was performed by means of whole-cell patch-clamp recordings of Cav1.2 Ca2+ [ICa(L)] or Ba2+ [IBa(L)] current as well as KCa1.1 currents in single myocytes and by measuring contractile responses in endothelium-denuded isolated rings. At a holding potential (Vh) of −80 mV, cardamonin decreased both IBa(L) and ICa(L) in a concentration-dependent manner with similar pIC50 values. The maximum of the IBa(L)-voltage relationship was shifted by 10 mV in the hyperpolarizing direction, but threshold remained unaffected. Cardamonin modified both the activation and the inactivation kinetics of IBa(L) and shifted the voltage dependence of both inactivation and activation curves to more negative potentials by 19 and 7 mV, respectively, thus markedly decreasing the Ba2+ window current. Block of IBa(L) was frequency-dependent, and rate of recovery from inactivation was slowed. Cardamonin increased KCa1.1 currents in a concentration-dependent manner; this stimulation was iberiotoxin- and BAPTA [1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid]-sensitive. On the contrary, iberiotoxin did not modify cardamonin-induced relaxation of rings precontracted either with phenylephrine or with (S)-(−)-methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)pyridine-5-carboxylate [(S)-(−)-Bay K 8644]. The overall effects of cardamonin were incompletely reversed by washout. In conclusion, cardamonin is a naturally occurring, bifunctional vasodilator that, by simultaneously inhibiting ICa(L) and stimulating KCa1.1 current, may represent a scaffold for the design of novel drugs of potential interest for treatment of systemic hypertension.