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Norbormide [5-(α-hydroxy-α-2-pyridylbenzyl)-7-(α-2-pyridylbenzylidene)-5-norbornene-2,3-dicarboximide] (NRB, 1), an existing but infrequently used rodenticide, is known to be uniquely toxic to rats, but relatively harmless to other rodents and mammals. As a vasoactive agent, NRB induces a species-specific vasocontractile effect that is restricted to the peripheral arteries of the rat. Despite the precise mechanisms behind this phenomenon having yet to be fully clarified, it is postulated that the molecular target of NRB could be located within the plasma membrane of rat peripheral artery myocytes (e.g. rat caudal artery myocytes). As such, the primary objective of this study was to develop a fluorescently labelled derivative of NRB to investigate its subcellular distribution/localization in both NRB-sensitive (freshly isolated rat caudal artery myocytes, FIRCAMs) and NRB-insensitive (human hepatic stellate, LX2) cells. Of the examples prepared, lead structure endo-NRB-NBD-bPA subsequently demonstrated retention of the parent toxicant's pharmacological profile (in terms of its ability to induce both a vasocontractile response in rat caudal artery rings in vitro, and a lethal end-point in rats in vivo). Endo-NRB-NBD-bPA was also shown to be significantly less permeable (an integral feature in the design of fluorescent probes targeting cell-surface receptors) to both LX2 cells and FIRCAMs. Disappointingly, no fluorescence could be observed on the plasma membrane of FIRCAMs stained with endo-NRB-NBD-bPA.

Chronically administered organic nitrates induce nitrate tolerance and endothelial dysfunction, which limit their therapeutic use. eNOS uncoupling, ROS over-production, aldehyde dehydrogenase-2 as well as superoxide dismutase (SOD) oxidative inhibition, and cGMP desensitization are thought to play an important role. Natural polyphenols are effective antioxidants, which might counteract the mechanisms leading to nitrate tolerance. The aim of this work was to verify whether freeze-dried (dealcoholized) red wine (FDRW) was able to revert glyceryl trinitrate (GTN) tolerance and endothelial dysfunction induced in rat aorta rings with either GTN or diethyldithiocarbamate (DETCA), an irreversible inhibitor of Cu/Zn SOD. GTN induced a concentration-dependent relaxation of rings pre-contracted with phenylephrine. GTN spasmolysis was significantly reduced in rings pre-incubated with either GTN or DETCA. FDRW, at 2.8 µg of gallic acid equivalents (GAE)/mL concentration, was able to revert partially, though significantly, GTN-induced tolerance but not tolerance and endothelial dysfunction induced by DETCA. This work provides the first evidence in vitro that red wine components, at concentrations comparable to those achieved in human blood after moderate consumption of red wine, revert tolerance to nitrates with a mechanism possibly mediated by SOD.

In the search for novel chemical scaffolds leading to potential antihypertensive agents, the methanol extract of Murraya paniculata leaves was assessed for its effects on isolated rat aorta rings. The vasorelaxing effect of the chloroform fraction of the methanol plant extract was the most potent for its vasorelaxing activity on rat aorta rings contracted by 60 mM K(+) (K60). Two coumarins were isolated from the chloroform fraction: the novel kimcuongin (1) and the known murracarpin (2). Their structures were determined from spectroscopic evidences including (1)H- and (13)C-NMR, correlation spectroscopy (COSY), nuclear Overhauser effect spectroscopy (NOESY), heteronuclear multiple bond correlation (HMBC), heteronuclear single quantum correlation (HSQC), and high resolution mass spectrometry (HR-MS). Kimcuongin and, to a lesser extent, murracarpin, showed vasorelaxing activity with IC50 values of 37.7 µM and 139.3 µM, respectively. The coumarins kimcuongin and murracarpin may thus represent a novel class of vasodilators of natural source.

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.Twenty-four flavonoids were analysed for their effects on I(Ca1.2) in rat tail artery myocytes, using the whole-cell patch-clamp method.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).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.

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 x 10(-7) and 2.23 x 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 x 10(-6) and 6.90 x 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 microm 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 x 10(-7) and 4.27 x 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. British Journal of Pharmacology (2004) 141, 415-422. doi:10.1038/sj.bjp.0705635

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 Ca(2+) channels.We investigated the mechanical and electrophysiological properties of quercetin and its rutoside, rutin, in an attempt to clarify how Ca(2+) channel activation might be related to the myorelaxing activity.Aorta ring preparations and single tail artery myocytes were employed for functional and patch-clamp experiments, respectively.Rutin was found to relax intact rat aorta rings, which had been precontracted with phenylephrine (pIC(50) = 5.65 +/- 0.31) 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 Ca(2+) 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 pIC(50), 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 pIC(50) of 4.59 +/- 0.03. The application of quercetin in isolated smooth muscle cells brought about a marked increase of L-type Ca(2+) current (pEC(50) = 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 microM quercetin. The application of Bay K 8644 (10-100 nM) caused a further significant increase in L-type Ca(2+) current in rat tail artery myocytes stimulated with 30 microM quercetin.Quercetin is a naturally occurring L-type Ca(2+) channel agonist. This effect, however, is overwhelmed by quercetin-induced vasorelaxation taking place via pathways which are more relevant than L-type Ca(2+) influx in the hierarchy of functional competencies.

1. 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.

1 Aim of the present study was to investigate the effects of norbormide, a selective vasoconstrictor agent of the rat peripheral vessels, on the whole-cell voltage-dependent L-type Ca(2+) current (I(Ca(L))) of freshly isolated smooth muscle cells from the rat caudal artery, using either the conventional or the amphotericin B-perforated whole-cell patch-clamp method. 2 Norbormide decreased L-type Ca(2+) current in a concentration- and voltage-dependent manner, without modifying the threshold and the maximum of the current-voltage relationship. Norbormide-induced I(Ca(L)) inhibition was reversible upon wash-out. 3 Norbormide both shifted the voltage dependence of the steady-state inactivation curve to more negative potentials by about 16 mV, without affecting the activation curve, and decreased the slope of inactivation. Norbormide, however, did not modify both the activation and the inactivation kinetics of the I(Ca(L)). 4 Norbormide decreased I(Ca(L)) progressively during repetitive step depolarizations, with inhibition depending on the stimulation frequency (use-dependent block) as well as on the holding potential. 5 Addition of 50 micro M norbormide caused the contraction of all freshly isolated cells and also of those impaled with the perforated method, but not of those impaled with the conventional method (i.e. dialysed). 6 In conclusion, these results prove norbormide to be a vascular L-type Ca(2+) channel inhibitor, which preferentially acts on the inactivated and/or open state of the channel. In rat caudal artery smooth muscle, however, this mechanism does not result in a vasodilating effect since it is overwhelmed by the mechanism underlying norbormide-induced vasoconstriction.

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.

Therapies addressing multiple stages of Plasmodium falciparum life cycle are highly desirable for implementing malaria elimination strategies. MMV019918 (1, 1-[5-(4-bromo-2-chlorophenyl)furan-2-yl]-N-[(piperidin-4-yl)methyl]methanamine) was selected from the MMV Malaria Box for its dual activity against both asexual stages and gametocytes. In-depth structure-activity relationship studies and cytotoxicity evaluation led to the selection of 25 for further biological investigation. The potential transmission blocking activity of 25 versus P. falciparum was confirmed through the standard membrane-feeding assay. Both 1 and 25 significantly prolonged atrioventricular conduction time in Langendorff-isolated rat hearts, and showed inhibitory activity of Ba(2+) current through Ca(v)1.2 channels. An in silico target-fishing study suggested the enzyme phosphoethanolamine methyltransferase (PfPMT) as a potential target. However, compound activity against PfPMT did not track with the antiplasmodial activity, suggesting the latter activity relies on a different molecular target. Nevertheless, 25 showed interesting activity against PfPMT, which could be an important starting point for the identification of more potent inhibitors active against both sexual and asexual stages of the parasite.

We present a series of oxadiazolothiazinones, selective inotropic agents on isolated cardiac tissues, devoid of chronotropy and vasorelaxant activity. Functional and binding data for the precursor of the series (compound 1) let us hypothesize LTCC blocking activity and the existence of a recognition site specific for this scaffold. We synthesized and tested 22 new derivatives: introducing a para-methoxyphenyl at C-8 led to compound 12 (EC50 = 0.022 μM), twice as potent as its para-bromo analogue (1). For 10 analogues, we extended the characterization of the biological properties by including the assessment of metabolic stability in human liver microsomes and cytochrome P450 inhibition potential. We observed that the methoxy group led to active compounds with low metabolic stability and high CYP inhibition, whereas the protective effect of bromine resulted in enhanced metabolic stability and reduced CYP inhibition. Thus, we identified two para-bromo benzothiazino-analogues as candidates for further studies.