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CaV1.2 channel blockers or 5-HT2 receptor antagonists constitute effective therapy for Raynaud’s syndrome. A functional link between the inhibition of 5-HT2 receptors and CaV1.2 channel blockade in arterial smooth muscles has been hypothesized. Therefore, the effects of ritanserin, a nonselective 5-HT2 receptor antagonist, on vascular CaV1.2 channels were investigated through electrophysiological, functional, and computational studies. Ritanserin blocked CaV1.2 channel currents (ICa1.2) in a concentration- dependent manner (Kr = 3.61 μM); ICa1.2 inhibition was antagonized by Bay K 8644 and partially reverted upon washout. Conversely, the ritanserin analog ketanserin (100 μM) inhibited ICa1.2 by ~50%. Ritanserin concentration-dependently shifted the voltage dependence of the steady-state inactivation curve to more negative potentials (Ki = 1.58 μM) without affecting the slope of inactivation and the activation curve, and decreased ICa1.2 progressively during repetitive (1 Hz) step depolarizations (use- dependent block). The addition of ritanserin caused the contraction of single myocytes not yet dialyzed with the conventional method. Furthermore, in depolarized rings, ritanserin, and to a lesser extent, ketanserin, caused a concentration-dependent relaxation, which was antagonized by Bay K 8644. Ritanserin and ketanserin were docked at a region of the CaV1.2 α1C subunit nearby that of Bay K 8644; however, only ritanserin and Bay K 8644 formed a hydrogen bond with key residue Tyr-1489. In conclusion, ritanserin caused in vitro vasodilation, accomplished through the blockade of CaV1.2 channels, which was achieved preferentially in the inactivated and/or resting state of the channel. This novel activity encourages the development of ritanserin derivatives for their potential use in the treatment of Raynaud’s syndrome.

Quercetin, a flavonoid abundantly present in the Mediterranean diet, is considered a vasodilator despite its recognized capability to stimulate vascular Ca

The cardiac action potential is regulated by several ion channels. Drugs capable to block these channels, in particular the human ether-a-go-go-related gene (hERG) channel, also known as KV11.1 channel, may lead to a potentially lethal ventricular tachyarrhythmia called "Torsades de Pointes". Thus, evaluation of the hERG channel off-target activity of novel chemical entities is nowadays required to safeguard patients as well as to avoid attrition in drug development. Flavonoids, a large class of natural compounds abundantly present in food, beverages, herbal medicines, and dietary food supplements, generally escape this assessment, though consumed in consistent amounts. Continuously growing evidence indicates that these compounds may interact with the hERG channel and block it. The present review, by examining numerous studies, summarizes the state-of-the-art in this field, describing the most significant examples of direct and indirect inhibition of the hERG channel current operated by flavonoids. A description of the molecular interactions between a few of these natural molecules and the Rattus norvegicus channel protein, achieved by an in silico approach, is also presented.

To characterize the role of cAMP-dependent protein kinase (PKA) in regulating vascular Ca2+ current through Cav1.2 channels [ICa1.2], we have documented a marked capacity of the isoquinoline H-89, widely used as a PKA inhibitor, to reduce current amplitude. We hypothesized that the ICa1.2 inhibitory activity of H-89 was mediated by mechanisms unrelated to PKA inhibition. To support this, an in-depth analysis of H-89 vascular effects on both ICa1.2 and contractility was undertaken by performing whole-cell patch-clamp recordings and functional experiments in rat tail main artery single myocytes and rings, respectively. H-89 inhibited ICa1.2 with a pIC50 (M) value of about 5.5, even under conditions where PKA activity was either abolished by both the PKA antagonists KT5720 and protein kinase inhibitor fragment 6-22 amide or enhanced by the PKA stimulators 6-Bnz-cAMP and 8-Br-cAMP. Inhibition of ICa1.2 by H-89 appeared almost irreversible upon washout, was charge carrier- and voltage-dependent, and antagonised by the Cav1.2 channel agonist (S)-(-)-Bay K 8644. H-89 did not alter both potency and efficacy of verapamil, did not affect current kinetics or voltage-dependent activation, while shifting to the left the 50% voltage of inactivation in a concentration-dependent manner. H-89 docked at the α1C subunit in a pocket region close to that of (S)-(-)-Bay K 8644 docking, forming a hydrogen bond with the same, key amino acid residue Tyr-1489. Finally, both high K+- and (S)-(-)-Bay K 8644-induced contractions of rings were fully reverted by H-89. In conclusion, these results indicate that H-89 inhibited vascular ICa1.2 and, consequently, the contractile function through a PKA-independent mechanism. Therefore, caution is recommended when interpreting experiments where H-89 is used to inhibit vascular smooth muscle PKA.

Dalbergia species heartwood, widely used in traditional medicine to treat various cardiovascular diseases, might represent a rich source of vasoactive agents. In Vietnam, Dalbergia tonkinensis is an endemic tree. Therefore, the aim of the present work was to investigate the vascular activity of R-(−)-3′-hydroxy-2,4,5-trimethoxydalbergiquinol isolated from the heartwood of D. tonkinensis and to provide circular dichroism features of its R absolute configuration. The vascular effects of R-(−)-3′-hydroxy-2,4,5-trimethoxydalbergiquinol were assessed on the in vitro mechanical activity of rat aorta rings, under isometric conditions, and on whole-cell Ba2+ currents through CaV1.2 channels (IBa1.2) recorded in single, rat tail main artery myocytes by means of the patch-clamp technique. R-(−)-3′-Hydroxy-2,4,5-trimethoxydalbergiquinol showed concentration-dependent, vasorelaxant activity on both endothelium-deprived and endothelium intact rings precontracted with the α 1 receptor agonist phenylephrine. Neither the NO (nitric oxide) synthase inhibitor Nω-nitro-L-arginine methyl ester nor the cyclooxygenase inhibitor indomethacin affected its spasmolytic activity. R-(−)-3′-Hydroxy-2,4,5-trimethoxydalbergiquinol-induced vasorelaxation was antagonized by (S)-(−)-Bay K 8644 and unaffected by tetraethylammonium plus glibenclamide. In patch-clamp experiments, R-(−)-3′-hydroxy-2,4,5-trimethoxydalbergiquinol inhibited IBa1.2 in a concentration-dependent manner and significantly decreased the time constant of current inactivation. R-(−)-3′-Hydroxy-2,4,5-trimethoxydalbergiquinol likely stabilized the channel in its closed state, as suggested by molecular modelling and docking simulation to the CaV1.2 channel α 1c subunit. In conclusion, D. tonkinensis species may represent a source of agents potentially useful for the development of novel antihypertensive drugs.

3,5-Di-t-butyl-4-hydroxyanisole (DTBHA) is considered as an activator of the skeletal muscle sarcoplasmic reticulum (SR) Ca

This study was designed to unveil effects of 5-nm sized, polyvinylpyrrolidone-coated gold nanoparticles (AuNPs) on vascular CaV1.2 and CaV3.1 channels. Ba2+ currents through both channels (IBa1.2 and IBa3.1) were recorded in single myocytes isolated from the rat tail main artery by means of the whole-cell patch-clamp method. AuNPs increased IBa1.2 and IBa3.1 amplitude in a concentration- and Vh-dependent manner. Neither the voltage dependence of inactivation and activation curves nor inactivation and activation kinetics were affected by AuNPs. In conclusion, these findings warrant further investigation to clarify whether different types of NPs possess the same stimulatory activity and may represent a toxic hazard to humans.

3,5-Di-t-butyl-4-hydroxyanisole (DTBHA) is considered as an activator of the skeletal muscle sarcoplasmic reticulum (SR) Ca2+-uptake, endowed with antioxidant and L-type Ca2+ channel blocking activities. In this study we assessed the cardiac effects of DTBHA on Langendorff perfused rat hearts, isolated rat atria and rat cardiac SR membrane vesicles, as well as on several SERCA isoforms of membrane preparations. Moreover, in order to clarify its molecular mechanism of action Ca2+ imaging experiments were carried out on HEK293 cells transiently transfected with RyR2 channel. Docking of DTBHA at the rat RyR2 protein was investigated in silico. In Langendorff perfused rat hearts, DTBHA significantly increased, in a concentration-dependent manner, left ventricular pressure and diastole duration, while reducing heart rate and the time-constant of isovolumic relaxation, leaving unaltered coronary perfusion pressure. At the maximum concentration tested (30 µM), it significantly prolonged PQ interval, but left the corrected QT intervals unaffected. In spontaneously beating atria, DTBHA decreased sinus rate in a concentration-dependent manner. DTBHA, at concentrations higher than 10 µM, increased Ca2+ uptake in cardiac SR without affecting Ca2+-dependent ATPase activity assayed on several SERCA isoforms. Moreover, DTBHA antagonized thapsigargin-stimulated Ca2+ leak in cardiac SR and reduced caffeine-induced, RyR2-activated Ca2+ release in RyR2 expressing HEK293 cells. Using computational approaches, DTBHA showed a good affinity outline into binding sites of RyR2 protein. In conclusion, DTBHA behaved like a negative chronotropic, a positive inotropic and a lusitropic agent on rat heart preparations and improved cardiac SR Ca2+ uptake by lowering SR Ca2+ leak.

This study was designed to unveil effects of 5-nm sized, polyvinylpyrrolidone-coated gold nanoparticles (AuNPs) on vascular Ca

To characterize the role of cAMP-dependent protein kinase (PKA) in regulating vascular Ca

In the search for novel natural compounds endowed with potential antihypertensive activity, a new sulfur-containing indole alkaloid, N-demethylglypetelotine (2), and its known analogue glypetelotine (1), were isolated from the leaves of Glycosmis petelotii. Their structures were established on the basis of spectroscopic evidence. The two alkaloids were assessed for vasorelaxing activity on rat aorta rings and for L-type Ba2+ current [IBa(L)] blocking activity on single myocytes isolated from rat tail artery. Both glypetelotine and N-demethylglypetelotine inhibited phenylephrine-induced contraction with IC50 values of 20 and 50 μM, respectively. The presence of endothelium did not modify their spasmolytic effect. Neither glypetelotine nor N-demethylglypetelotine affected Ca2+ release from the sarcoplasmic reticulum induced by phenylephrine. The spasmolytic effect of glypetelotine increased with membrane depolarization. In the presence of 60 mM K+, both compounds inhibited, in a concentration-dependent mann...

Ion 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 Ca

Cannabinoid CB2 receptor activation has been shown to have many pharmacological but not psychotropic effects. The aim of this study was to investigate the potential protection of brain tissues afforded by the novel substituted 4-quinolone-3-carboxylic acid derivative COR167, a selective CB2 agonist, toward ischemia and reperfusion-induced injury, as well as the mechanism of this potential effect. Rat brain cortical slices subjected to oxygen and glucose deprivation (OGD) followed by re-oxygenation were used. Cell damage was quantified by measuring at the end of the reperfusion phase the release into the artificial cerebrospinal fluid (ACSF) of lactate dehydrogenase (LDH), glutamate, IL-6 and TNF-α and by evaluating in tissue the lipid-peroxides (thiobarbituric acid-reactive substances, TBARS), the free, reduced glutathione content (GSH) and the water gain (TWG), taken as an index of cell swelling. COR167 (10nM or 100 nM), added to ACSF during the entire reperfusion phase, markedly reduced LDH and glutamate release, as well as TWG. Lower (0.1-1 nM) or higher concentrations (1,000 nM) were ineffective, suggesting thereby an hormetic behavior. COR167 at 10nM concentration markedly reverted in tissues TBARS increase and GSH decrease, while reducing IL-6 and TNF-α release into ACSF. COR167 effects on glutamate and LDH release were abrogated by the selective CB2 inverse-agonists COR170 (1 nM) and AM630 (1μM) but not by the CB1 antagonist AM251 (1 μM). COR170 as well as AM630 per se were able to revert TWG. The CB2 receptor agonist COR167 potently protected rat brain cortical slices against OGD and reperfusion injury, partly through CB2 receptors activation.

Regulation of smooth muscle contraction is driven by a number of protein kinases: the evidence for this often originates from studies that investigate the effects of extracellularly added specific protein kinase inhibitors. Six compounds, thought to be selective inhibitors of various kinases, were analysed for their effects on vascular L-type Ca(2+) channels because this potential subsidiary activity could strongly influence our understanding of the pathways involved in smooth muscle contraction. Whole-cell L-type Ba(2+) currents [I(Ba(L))] were recorded in single myocytes, and contractile responses were measured from endothelium-denuded rings taken from the rat tail artery. Although ML-7, ML-9, and wortmannin (MLCK inhibitors), HA-1077 and Y-27632 (Rho-associated kinase inhibitors), and GF-109203X (PKC inhibitor) relaxed rings pre-contracted with high KCl in a concentration-dependent manner, their effect on I(Ba(L)) intensity was surprisingly variable. Wortmannin showed negligible effects while HA-1077 and Y-27632 were ineffective. I(Ba(L)) was partly inhibited by GF-109203X and blocked by ML-7 and ML-9 in a concentration-dependent manner, with the blockade by ML-7 being voltage-dependent. Whilst ML-7, ML-9, and GF-109203X sped up the inactivation kinetics of I(Ba(L)), GF-109203X did not modify ML-7- or ML-9-induced effects, with both intensity and kinetics of the current remaining unchanged. In contrast, application of Bay K 8644 on myocytes pre-treated with ML-7 or ML-9 raised I(Ba(L)) beyond control values. In conclusion, ML-7 and ML-9 inhibit L-type Ca(2+) channels via a mechanism independent of MLCK, PKC or Rho kinase activities, and as such caution should be used in employing these agents to elucidate the role of kinases in smooth muscle contraction.

The aim of this study was to investigate the effects of the four isomers (3a, 3b, 3c and 3d) of a novel multidrug resistance-reverting agent – 3,4,5-trimethoxybenzoic acid 4-(methyl-{4-[3-(3,4,5-trimethoxyphenyl)acryloyloxy]cyclohexyl}amino)cyclohexyl ester – on vascular functions in vitro. A comparison of their mechanical and electrophysiological actions in rat aorta rings and single rat tail artery myocytes, respectively, was performed. In rat aorta rings, 3a–d antagonized both 60 mmol/l K+- and phenylephrine-induced contraction in a concentration-dependent manner, with maximal relaxation values averaging 50% of controls, 3d being the most effective of the series. The vasorelaxing effect was similar either in presence or absence of intact endothelium. In rat tail artery myocytes, out of the four isomers, only 3a consistently inhibited Ba2+ current through Cav1.2 channels. Our results provide functional evidence that 3a–d are weak vasorelaxing agents, although at concentrations much higher than those effective for multidrug resistance reversion in cancer cells.

The aim of this study was to investigate the effects of 3,5-dibenzoyl-4-(3-phenoxy-phenyl)-1,4-dihydro-2,6-dimethylpyridine (DP7), a novel multidrug resistance (MDR) reverter, on cytochrome P450 (CYP)-activities by human and rat liver microsomes. Effects of DP7 were assessed with use of selective substrates, markers of CYP activities. With rat microsomes, ethoxyresorufin (ETR) was used as substrate for CYP1A1, penthoxyresorufin (PTR) for 2B, benzyloxyresorufin (BZR) for 1A1/2, 2B, 2C, 3A. CYP3A enzyme activities of rat (3A2) and human (3A4) liver microsomes, were assessed fluorimetrically using either 7-benzyloxy-quinoline (BQ) or [3-[3(3,4-difluorobenzyl)oxy]-5,5-dimethyl-4-[4-(methylsulfonyl)-phenyl]furan-2-(5H)-one] (DFB). When rat microsomes were incubated with DP7, concentration-inhibition curves were obtained. DP7 inhibitions gave IC(50) values of 3.8 microM for PTR, 3.8 microM for ETR and 10.4 microM for BZR and were not competitive in nature; moreover, they were reversible. When BQ was used as substrate of rat microsomes, DP7 inhibited its oxidation with an IC(50) value of 4.17 microM, while this oxidation was inhibited by only 25% at the highest DP7 concentration used (75 microM) with human microsomes. On the contrary, when DFB was used as substrate, DP7 showed identical IC(50) values (34.67 microM) with microsomal preparations from either species. The moderate inhibition of CYP isoforms of rat liver microsomes and the weak inhibition of human CYP3A4 enzyme activity operated by DP7, suggest that DP7 in man should not give rise to important, unpredictable pharmacokinetic interactions. This conclusion supports the role of this compound as a lead for the development of novel MDR reverterting dihydropyridines of therapeutic interest.

The aim of this study was to investigate the effect of 17β-oestradiol (E2) on detrusor smooth muscle contractility and its possible neuroprotective role against ischaemic-like condition, which could arise during overactive bladder disease. The effect of E2 was investigated on rat detrusor muscle strips stimulated with carbachol, KCl and electrically, in the absence or presence of a selective oestrogen receptor antagonist (ICI 182,780) and, by using confocal Ca2+ imaging technique, measuring the amplitude (ΔF/F0) and the frequency of spontaneous whole cell Ca2+ flashes. Moreover, the effect of 1 and 2 h of anoxia–glucopenia and reperfusion (A-G/R), in the absence or presence of the hormone, was evaluated in rat detrusor strips perfused with Krebs solution which underwent electrical field stimulation to stimulate intrinsic nerves; the amplitude and the frequency of Ca2+ flashes were also measured. 17β-Oestradiol exhibited antispasmogenic activity assessed on detrusor strips depolarized with 60 mm KCl at two different Ca2+ concentrations. 17β-Oestradiol at the highest concentration tested (30 μm) significantly decreased detrusor contractions induced by all the stimuli applied. In addition, the amplitude and the frequency of spontaneous Ca2+ flashes were significantly decreased in the presence of E2 (10 and 30 μm) compared with control detrusor strips. In strips subjected to A-G/R, a significant increase in the amplitude of both spontaneous and evoked flashes was observed. 17β-Oestradiol was found to increase the recovery of detrusor strips subjected to A-G/R. The ability of E2 to suppress contraction in control conditions may explain its ability to aid recovery following A-G/R.

Aim To study the effect of 17β-estradiol (E2) as a neuroprotective agent in male and female rat urinary bladders subjected to Anoxia-Glucopenia and Reperfusion (A/G-R) damage. Methods Rat urinary bladders were exposed to 1 hour of A—G and 2 hours of reperfusion (R). Intrinsic nerves underwent electrical field stimulation. The effect of E2 on the contractile response and its recovery in R phase, was monitored by adding it to the bath medium, at different concentrations, 60 min before A—G, during the A—G and the first 30 min of R. Furthermore, on both genders, in vivo treatments with E2, testosterone, IC1 182,780 and anastrozole were performed. Results After A—G the recovery of the nerve function in female bladders were significantly higher than in male. E2 given both in vivo (increased of E2 plasma levels were monitored) and in vitro, resulted to be neuroprotective in male bladder subjected to A—G/R damage. When rats were treated with anastrozole, a lower recovery of nerve responses both in male and female bladders was observed. Finally, treatments with E2 receptor antagonist ICI 182,780 increased E2 plasma levels and showed to abolished E2 neuroprotection, thus suggesting that E2 promotes nelronal survival likely through a rapid estrogen receptor mediated response. Conclusions Male rat urinary bladder serves are more susceptible to A—G/R damage than female. E2 exhibits neuroprotective properties and could be a lead for novel agents capable to protect the urinary bladder from ischaemia/reperfusion damage associated with urinary bladder disorders. Neurourol. Urodyn. 28:535–541, 2009. © 2008 Wiley-Liss, Inc.

The vasorelaxing effect of isolates (compounds 1, 2, 3, and 4 (homoisoflavanones), compound 5 (sesquiterpenoid), compounds 6 and 7 (bufadienolides)) from the South African Hyacinthaceae has been assessed using rat aortic ring preparations. Compounds 2, 3, and 4 inhibited the tonic contraction induced by both 60 mm K+ (K60) and phenylephrine, compound 3 being the most potent. Compounds 5, 6, and 7 caused a modest concentration-dependent relaxation, whereas compound 1 was ineffective. Under K25- or K60-induced depolarization, compound 3 displayed antispasmodic effects not reversed by tetraethylammonium. Under precontraction induced with phenylephrine, compound 3 shifted to the left the concentration-relaxation curves of either isoprenaline or sodium nitroprusside. 1 H-[1,2,4] oxidazolol [4,3-a] quinoxalin-1-one shifted to the right the concentration-relaxation curve of compound 3, while 3′-isobutyl-1-methylxanthine had no effect. In the absence of extracellular Ca2+, compound 3 (estimated pIC50 = 4.66) and ryanodine reduced the response to phenylephrine. Phenylephrine-stimulated influx of extracellular Ca2+ was markedly reduced when tissues were pretreated with compound 3 (pIC50 = 5.14) or nifedipine, but stimulated by ryanodine. Compound 3 partially antagonized the contraction induced by phorbol 12-myristate-13-acetate. To our knowledge, this has been the first account describing the vasodilating activity of homoisoflavonoids: compound 3 proved an effective vasorelaxing agent, partly acting via the activation of soluble guanylyl cyclase.

Papaverine 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 ≥ 30 μM, 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.

Rhodamine 123 (R123) transport substrate sensitizes P-glycoprotein (P-gp) to inhibition by compound 2c (cis-cis) N,N-bis(cyclohexanolamine)aryl ester isomer in a concentration-dependent manner in human MDR1-gene transfected mouse T-lymphoma L5178 cells as shown previously. By contrast, epirubicin (EPI) concentration changes left unaltered 2c IC50 values of EPI efflux. To clarify this discrepancy, defined molecular docking (DMD) analyses of 12 N,N-bis(cyclohexanolamine)aryl esters, the highly flexible aryl ester analog 4, and several P-gp substrate/non-substrate inhibitors were performed on human P-gp drug- or nucleotide-binding domains (DBD or NBD). DMD measurements yielded lowest binding energy (LBE, kcal/mol) values (mean ± SD) ranging from -11.8 ± 0.54 (valspodar) to -3.98 ± 0.01 (4). Lys234, Ser952 and Tyr953 residues formed H-bonds with most of the compounds. Only 2c docked also at ATP binding site (LBE value of -6.9 ± 0.30 kcal/mol). Inhibition of P-gp-mediated R123 efflux by 12 N,N-bis(cyclohexanolamine)aryl esters and 4 significantly correlated with LBE values. DMD analysis of EPI, (3)H-1EPI, (3)H-2EPI, (14)C-1EPI, (14)C-2EPI, R123 and 2c before and after previous docking of each of them indicated that pre-docking of either 2c or EPI significantly reduced LBE of both EPI and R123, and that of both (3)H-2EPI and (14)C-2EPI, respectively. Since the clusters of DBD amino acid residues interacting with EPI were different, if EPI docked alone or after pre-docking of EPI or 2c, the existence of alternative secondary binding site for EPI on P-gp is credible. In conclusion, 2c may allocate the drug-binding pocket and reduce strong binding of EPI and R123 in agreement with P-gp inhibition experiments, where 2c reduced efflux of EPI and R123.

Mycobacterium tuberculosis (M. tuberculosis), an almost genetically monomorphic pathogen is a human parasite, transmitted mostly by humans and causes tuberculosis (TB). TB is firmly associated to poverty, although lack of proper nutrition and lowered immune status are contributing factors for disease development. TB remains second only to HIV/AIDS as the leading cause of mortality worldwide due to a single infectious agent and is responsible for nearly 1.5 million deaths annually. Some steps of the progress of our knowledge of M. tuberculosis physiology and its interactions with human beings, are reviewed here. This progress has provided fertile ground for improving diagnosis and cure of TB infection. For TB diagnostics laboratories in high-burden countries, primary isolation is the first step before performing drug susceptibility testing (DST) of M. tuberculosis. IGRA (interferon-γ release assay)-based tests for diagnosis of active TB are sufficiently fast, specific and sensitive to allow to contain infection and distinguish among latent TB infection and BCG vaccination individuals from those who have clinically resolved M. tuberculosis infection after anti-TB treatment.

The great progress of knowledge of both M. tuberculosis physiology and how human host and bacilli interact has provided fertile ground for improving diagnosis and cure of TB infection. Once M. tuberculosis has infected humans, it elaborates strategies for evading the risk to killing by the cells of the host immune system and by the anti-tuberculosis (anti-TB) agents employed to cure infection. These strategies give rise to a bacterial multidrug resistance (MDR) status. This stems firstly from genetic mutations targeting a constellation of drug-processing mechanisms that still need full identification, as drug efflux pumps and drug activating/ inactivating enzymes (genetic resistance). Secondly, from the bacterial adaptation to stressful environmental conditions by adopting a temporary dormancy state lasting for decades and characterized by indifference to anti-TB drugs (phenotypic resistance or tolerance). The clarification of the strategies elaborated for surviving by M. tuberculosis has brought to the identification in the last few years of a number of mycobacterial molecular targets worth to exploitation for the development of novel and powerful anti-TB drugs. These targets include drug-efflux pump systems, considered partly responsible for genetic multi-drug resistance, and several enzymes and pump systems, as well, that sustain the metabolic adaptations of M. tuberculosis in the host and give rise to its phenotypic drug resistance.

The use of the antiepileptic drug, 4-aminobutyrate transaminase (GABA-T) inhibitor vigabatrin (VIGA), has been recently cautioned because it is associated to irreversible field defects from damage of the retina. Since novel GABA-T inhibitors might prove useful in epilepsy or other CNS pathologies as VIGA substitutes, the aim of the present investigation was to characterize the biochemical properties of some taurine analogues (TA) previously shown to act as GABA-T inhibitors. These include (+/-)piperidine-3-sulfonic acid (PSA), 2-aminoethylphosphonic acid (AEP), (+/-)2-acetylaminocyclohexane sulfonic acid (ATAHS) and 2-aminobenzenesulfonate (ANSA). Kinetic analysis of the activity of partially purified rabbit brain GABA-T in the presence of VIGA and TA showed that PSA and AEP caused a linear, mixed-type inhibition (Ki values 364 and 1010 microM, respectively), whereas VIGA, ANSA and ATAHS behaved like competitive inhibitors (Ki values 320, 434 and 598 microM, respectively). Among the compounds studied, only VIGA exerted a time-dependent, irreversible inhibition of the enzyme, with Ki and k(inact) values of 773 microM and 0.14 min(-1), respectively. Furthermore, the ability of VIGA and TA to enhance GABA-ergic transmission was assessed in rabbit brain cortical slices by NMR quantitative analysis. The results demonstrate that VIGA as well as all TA promoted a significant increase of GABA content. In conclusion, PSA, ANSA and ATAHS, reversible GABA-T inhibitors with Ki values close to that of VIGA, represent a new class of compounds, susceptible of therapeutic exploitation in many disorders associated with low levels of GABA in brain tissues.

Aims To investigate the effect of anoxia/glucopenia and re-superfusion on intrinsic nerves in the mammalian urinary bladder. Methods Strips of detrusor smooth muscle were dissected from monkey and human urinary bladder and mounted for tension recording in organ baths superfused with Krebs solution. Human, monkey, and guinea-pig urinary bladders were treated to evaluate glycogen contents by a biochemical method. Results Detrusor strips from both monkeys and humans had to be exposed to anoxia-glucopenia for up to 2–2.5 hr to observe a progressive decline in the response to electrical field stimulation (EFS) of the intrinsic nerves, at variance with guinea-pig detrusor strips. In contrast, the response to direct activation of the smooth muscle with carbachol remained almost unaltered. Incubation of human and monkey detrusor strips with 2-deoxyglucose (2-DG) during 1 hr anoxia-glucopenia, however, caused a marked damage to the intrinsic nerves. The glycogen contents of both human detrusor specimens and monkey urinary bladders were 2.0- and 1.4-fold higher, respectively, than that found in guinea-pig urinary bladder; furthermore, untreated monkey detrusor sections showed a greater number of glycogen granules as compared to those subjected to anoxia-glucopenia and re-superfusion. In guinea-pig and in monkey detrusor sections glycogen granules were found in smooth muscle cells but not in neurons of intramural ganglia. Conclusions A higher susceptibility of guinea-pig as compared to monkey and human nerves has been demonstrated; it is suggested that anaerobic glucose metabolism during anoxia-glucopenia is crucial for the functional recovery of detrusor intrinsic nerves from damage caused by anoxia-glucopenia and re-superfusion. © 2004 Wiley-Liss, Inc.

Chronic use of glyceryl trinitrate is limited by serious side effects, inter alia tolerance and endothelial dysfunction of coronary and resistance arteries. The natural flavonoid quercetin has been shown to counteract the development of glyceryl trinitrate tolerance in vitro. Two mitochondriotropic, 4-O-triphenylphosphoniumbutyl derivatives of quercetin (QTA-3BTPI and Q-3BTPI) were compared to quercetin for protection against glyceryl trinitrate-induced tolerance and endothelial dysfunction of isolated rat aorta rings. Both QTA-3BTPI and Q-3BTPI significantly counteracted the reduced vascular responsiveness to both glyceryl trinitrate and acetylcholine caused by prolonged exposure of the vessel to glyceryl trinitrate itself, their potency being much greater than that of quercetin. QTA-3BTPI, however, turned out to cause endothelial dysfunction per se. Since Q-3BTPI antagonized in vitro nitrate tolerance and endothelial dysfunction of vessels, this encourages assessing whether this effect is displayed also in vivo during long-term glyceryl trinitrate treatment.

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

1. In order to ascertain whether both GABA(A) and GABA(B), or only GABA(B) receptors, directly modulate thermoregulation in conscious rabbits, GABA(A)/GABA(B) 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. 2. GABA (48 micromol), nipecotic acid (50 nmol), THIP (60 nmol), muscimol (18 nmol) and baclofen (8 nmol) induced hypothermia (-deltaRTmax values of 1.70+/-0.1, 1.4+/-0.2, 1.0+/-0.4, 1.1+/-0.2 and 1.6+/-0.3 degrees C, respectively), accompanied by inhibition of GMB and ECoG synchronization. THIP increased ps at delta frequency band (1.1-3.3 Hz), while GABA, nipecotic acid, muscimol and baclofen did the same at both delta and (4.6-6.5 Hz) frequency bands. ECoG ps changes were concomitant or even preceded hypothermia. 3. Bicuculline (1.8 nmol) induced hyperthermia (deltaRTmax 1.2+/-0.5 degrees C) and slight excitation of GMB, while CGP35348 (1.2 micromol) did not affect RT nor GMB. Both compounds did not affect ECoG ps. 4. Bicuculline potentiated muscimol-induced hypothermia, inhibition of GMB and synchronization of ECoG, while CGP35348 fully antagonized these effects. 5. In conclusion, the present results, while confirming the prevailing role of GABA(B), also outline a direct involvement of GABA(A) 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.

The aim of the present investigation was to characterize the cytochrome P450 (CYP)-dependent metabolism of l-deprenyl by brain microsomal preparations obtained from two different animal models that have been extensively used in Parkinson’s disease studies, namely monkey (Cercopithecus aethiops) and C57BL/6 mouse. In monkey brain microsomal fractions, the apparent Km values for methamphetamine formation from l-deprenyl were 67.8 ± 1.0 and 72.0 ± 1.6 lM, in the cortex and striatum, respectively. Similarly, for nordeprenyl formation from l-deprenyl, Km values in cortex and striatum were 21.3 ± 3.2 and 27.3 ± 4.0 lM, respectively. Both metabolic pathways appear to be more efficient in the cortex than in the striatum as the Vmax for microsomal preparation was lower in the striatum for the formation of both metabolites. The formation rate of l-methamphetamine was up to one order of magnitude greater than that of nordeprenyl. Inhibition analysis of both pathways in monkey brain suggested that l-methamphetamine formation is catalysed by CYP2A and CYP3A, whereas only CYP3A appears to be involved in nordeprenyl formation. With microsomal preparations from whole brain of C57BL/6 mice, the only l-deprenyl metabolite that could be detected was methamphetamine and the Km and Vmax values were similar to those determined in monkey cortex (53.6 ± 2.9 lM and 33.9 ± 0.4 pmol/min/mg protein, respectively). 4-Methylpyrazole selectively inhibited methamphetamine formation, suggesting the involvement of CYP2E1. In conclusion, the present study indicates that l-deprenyl is effectively metabolised by CYP-dependent oxidases in the brain, giving rise mainly to the formation of methamphetamine, which has been suggested to play a role in the pharmacological effects of the parent drug. The results also demonstrate that there are differences between species in CYP-dependent metabolism of l-deprenyl.

We have investigated the effects of the rat-specific vasoconstrictor agent norbormide on the mechanical and electrophysiological properties of rat non-vascular smooth muscles. Norbormide (50 microM) did not affect the resting tone of urinary bladder, tracheal, and duodenal rings. In all tissues, KCl-induced concentration-response curves were shifted downward by norbormide (5 and 50 microM). In urinary bladder and tracheal rings, norbormide inhibited contractile responses to carbachol only at the higher concentration (50 microM). In single gastric fundus myocytes, 50 microM norbormide inhibited L-type Ca(2+) current (I(Ca(L))) by about 60%, neither affecting both activation and inactivation rates of the current nor the current-voltage curve along the voltage axis. Our results indicate that rat non-vascular smooth muscles are relaxed by norbormide with a mechanism likely involving a reduction of Ca(2+) entry through L-type Ca(2+) channels.

(1) 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 [(3)H]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. (2) 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. (3) Taurine (1.2 x 10(-6)-4.8 x 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 x 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 x 10(-7) mol caused death within 24 h after treatment. (4) CAHS (4.8 x 10(-5) mol) antagonized the hyperthermic effect induced by TAG (1.2 x 10(-6) mol), AEA (1.2 x 10(-8) mol) or ISE (4.8 x 10(-5) mol). (5) In conclusion, these findings may indicate the existence of a recognition site specific for taurine, responsible for its effects on thermoregulation.

1. The aim of this study was to find taurinergic compounds that do not interact with brain GABA ergic systems. 2. Washed synaptic membranes (SM) from whole rabbit brain were able to bind [(3)H]muscimol. Saturation experiments of the binding of [(3)H]GABA to GABA(B) receptors showed that SM possess two binding components; twice Triton X-100-treated SM contained 0.048 mmol endogenous taurine/kg protein and bound [(3)H]taurine in a saturable manner (K(d)=249.0+/-6.3 nM and B(max)=3.4+/-1.0 pmol mg(-1) prot). 3. 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 [(3)H]taurine binding (K(i)=0.13, 0.13, 13.5 and 4.0 micro M, respectively). These analogues did not interact with GABA(A) and GABA(B) receptors and did not affect taurine- and GABA-uptake systems and GABA-transaminase activity. 4. 3-Aminopropanesulfonic acid (OMO), beta-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 [(3)H]muscimol binding to GABA(A) receptors with different affinities (K(i)=0.013, 7.9, 24.6, 47.5, 52.0, 91.0, 47.5, 118.1 and 166.3 micro M, respectively). Taurine, 2-aminoethylphosphonic acid, DMT, TMT and OMO inhibited the binding of [(3)H]GABA to GABA(B) receptors with K(i)'s in the micro M range (0.8, 3.5, 4.4, 11.3 and 5.0, respectively). GES inhibited taurine uptake (IC(50)=3.72 micro M) and PSA GABA transaminase activity (IC(50)=103.0 micro M). 5. In conclusion, AEA, TAG, ISE and CAHS fulfill the criteria for taurinergic agents.

Background: 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 ± 0.31) 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 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.

The sesquiterpene zerumbone, isolated from the rhizome of Zingiber zerumbet Sm., besides its widespread use as a food flavouring and appetiser, is also recommended in traditional medicine for the treatment of several ailments. It has attracted great attention recently for its effective chemopreventive and therapeutic effects observed in various models of cancer. To assess the zerumbone safety profile, a pharmacology study designed to flag any potential adverse effect on vasculature was performed. Zerumbone was tested for vasorelaxing activity on rat aorta rings and for L-type Ba(2+) current blocking activity on single myocytes isolated from the rat-tail artery. The spasmolytic effect of zerumbone was more marked on rings stimulated with 60 mM than with 30 mM K(+) (IC50 values of 16 µM and 102 µM, respectively). In the presence of 60 mM K(+), zerumbone concentration-dependently inhibited the contraction induced by the cumulative additions of Ca(2+), this inhibition being inversely related to the Ca(2+) concentration. Phenylephrine-induced contraction was inhibited by the drug, though less efficiently and independently of the presence of an intact endothelium, without affecting Ca(2+) release from the intracellular stores. Zerumbone inhibited the L-type Ba(2+) current (estimated IC50 value of 458.7 µM) and accelerated the kinetics of current decay. In conclusion, zerumbone showed an overall weak in vitro vasodilating activity, partly attributable to the blocking of the L-type Ca(2+) channel, which does not seem to represent, however, a serious threat to its widespread use.

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 Ca2+ current (ICa(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. Norbormide decreased L-type Ca2+ current in a concentration- and voltage-dependent manner, without modifying the threshold and the maximum of the current-voltage relationship. Norbormide-induced ICa(L) inhibition was reversible upon wash-out. 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 ICa(L). Norbormide decreased ICa(L) progressively during repetitive step depolarizations, with inhibition depending on the stimulation frequency (use-dependent block) as well as on the holding potential. Addition of 50 μM 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). In conclusion, these results prove norbormide to be a vascular L-type Ca2+ 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. British Journal of Pharmacology (2002) 137, 323–328. doi:10.1038/sj.bjp.0704877

In the search for novel natural compounds endowed with potential antihypertensive activity, a new sulfur-containing indole alkaloid, N-demethylglypetelotine (2), and its known analogue glypetelotine (1), were isolated from the leaves of Glycosmis petelotii. Their structures were established on the basis of spectroscopic evidence. The two alkaloids were assessed for vasorelaxing activity on rat aorta rings and for L-type Ba(2+) current [I(Ba(L))] blocking activity on single myocytes isolated from rat tail artery. Both glypetelotine and N-demethylglypetelotine inhibited phenylephrine-induced contraction with IC50 values of 20 and 50 μM, respectively. The presence of endothelium did not modify their spasmolytic effect. Neither glypetelotine nor N-demethylglypetelotine affected Ca(2+) release from the sarcoplasmic reticulum induced by phenylephrine. The spasmolytic effect of glypetelotine increased with membrane depolarization. In the presence of 60 mM K(+), both compounds inhibited, in a concentration-dependent manner, the contraction induced by cumulative addition of Ca(2+), this inhibition being inversely related to Ca(2+) concentration. Glypetelotine and, less efficiently N-demethylglypetelotine, inhibited I(Ba(L)), the former compound also affecting I(Ba(L)) kinetics. In conclusion, glypetelotine is a novel vasorelaxing agent which antagonizes L-type Ca(2+) channels.

3,5-Di- t -butyl-4-hydroxyanisole (DTBHA) increased in a concentration-dependent manner (calculated p ec 50 = 4.55 ± 0.18 M) the oxalate-stimulated Ca 2+ -pumping rate of rat skeletal muscle sarcoplasmic reticulum (SR) vesicles. Kinetic analysis of this effect suggested that the activation of SR Ca 2+ -ATPase operated by (DTBHA) was of both mixed and non-competitive type with respect to ATP in the range of concentrations 0.1–0.5 mM and above 1 mM, respectively; furthermore, it was independent of the free Ca 2+ concentrations. This indicated that the enzyme activation took place through the acceleration of the enzyme-substrate complex breakdown. Moreover, it appeared that its target site was cyclopiazonic acid sensitive. The uncommon ability of (DTBHA) to upregulate SR Ca 2+ uptake is of interest in view of its possible use for treating pathological conditions characterised by cell Ca 2+ overload as well as genetic disorders where SR Ca 2+ homeostasis is altered.

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 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.

The aim of the present investigation was to compare mechanical responses to electrical field stimulation (EFS), as well as cholinergic and non-adrenergic, noncholinergic (NANC) neurotransmission in guinea-pig, rat, monkey and human detrusor muscle strips. Responses to EFS (0.05, 0.5 and 1 ms pulse duration, 50 V, 1-15 Hz) of guinea-pig, rat, monkey and human detrusor muscle strips were recorded isometrically before and after blockade of muscarinic receptors and/or P2-purinoreceptors, as well as after desensitisation of P2-purinoceptors or blockade of the nerve impulse propagation. Single pulses of 0.05 ms duration elicited responses, in either guinea-pig or rat detrusor strips, which were abolished by tetrodotoxin (TTX), thus suggesting their neurogenic nature. In monkey and human detrusor strips, however, the same single pulses were not sufficient to generate contractile responses. The response of either rat or guinea-pig strips to single pulses of 0.5 ms and 1 ms duration was mainly myogenic in nature. While in rat and guinea-pig strips the neurogenic response was only partly reduced in the presence of atropine, in monkey and human strips it was abolished. In the presence of atropine, while suramin only partially reduced the EFS response either in rat or guinea-pig detrusor strips, a complete alpha,beta-methyleneATP-sensitive response was evident in guinea-pig detrusor strips. This suggests the involvement of other transmitter(s) beyond ATP in the NANC response of rat detrusor strips.

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 Ca2+ current (ICa) of freshly isolated smooth muscle cells from the guinea-pig gastric fundus, in the presence of a range of Ca2+ concentrations (1 – 5 mM) using the patch-clamp technique. The influx of Ca2+ had characteristics of L-type ICa (ICa(L)). BHA as well as DTBHA inhibited ICa(L) in a concentration-dependent manner, during depolarization to 10 mV from a holding potential of −50 mV. Bath application of BHA (50 μM) and DTBHA (30 μM) decreased ICa(L) by 48.9% and 45.2%, respectively. This inhibition was only partially reversible. In contrast, DIBHA (up to 50 μM) was devoided of effects on ICa(L). BHA inhibition of ICa(L) was voltage-dependent and inversely related to the external concentration of Ca2+. On the other hand, DTBHA inhibition was only voltage-dependent. 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. We conclude that the inhibition of ICa(L) by BHA and DTBHA is qualitatively similar to that of a Ca2+ channel blocker and is characterized by the stabilizing effect of the inactivated state of the channel. British Journal of Pharmacology (2001) 132, 1326–1332; doi:10.1038/sj.bjp.0703935

The neuroprotective agent riluzole [2-amino-6-(trifluoromethoxy)benzothiazole] has been shown to antagonize neuronal high-voltage activated Ca 2+ currents. In the search for novel scaffolds leading to potential antihypertensive agents, a series of 2-aryl- and 2-amido-benzothiazoles (HUP) were assessed for their vasorelaxing property on rat aorta rings and for their L-type Ba 2+ currents [ I Ba(L) ] blocking activity on single myocytes isolated from the rat tail artery. HUP5 and HUP30, the most potent of the series, inhibited phenylephrine-induced contraction with IC 50 values in the range 3–6 µM. The presence of endothelium did not modify their spasmolytic activity. Both HUP5 and HUP30 increased tissue levels of cGMP and shifted to the left the concentration–response curve to sodium nitroprusside. In rings precontracted by phenylephrine, tetraethylammonium or 1 H -[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (ODQ) shifted to the right the concentration–relaxation curves of HUP5 and HUP30. The antispasmodic effect of HUP5 and HUP30 was more marked on rings stimulated with 25/30 mM than with 60 mM K + . HUP5 and HUP30 antagonized both extracellular Ca 2+ influx and Ca 2+ mobilization from intracellular stores in response to phenylephrine: this effect was not modified by the presence of ODQ. I Ba(L) was partly inhibited by HUP5 and blocked by HUP30 in a concentration-dependent as well as ODQ-independent manner. In conclusion, HUP5 and HUP30 are vasorelaxing agents that stimulate soluble guanylyl cyclase, activate K + channels, and block extracellular Ca 2+ influx. The present benzothiazole derivatives form a novel class of multifunctional vasodilators which may give rise to effective antihypertensive agents.

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 present study was aimed at assessing the effects of either red blood cells (RBC) or RBC cross-linked with the bifunctional dimethyl suberimidate reagent (C-RBC) on contractile force (CFo), heart rate (HR) and coronary flow (CF) of the isolated rabbit heart hypoperfused with RBC suspensions under 30 mm Hg constant pressure. RBC or C-RBC caused a rapid and marked reduction of CF, CFo and HR. In RBC-treated hearts, however, reperfusion with Tyrode solution partially restored the initial myocardial parameters, while in C-RBC-treated hearts a rapid impairment of diastolic relaxation with a subsequent, steady and increasing heart contracture was observed. Histological analysis showed that in C-RBC-perfused hearts either capillaries or precapillary arterioles were occluded by C-RBC in spite of extensive washings with Tyrode solution. These findings indicate that C-RBC impair coronary circulation markedly and irreversibly.

3,5-Di- t -butyl catechol (DTCAT) releases Ca 2+ from rat skeletal muscle sarcoplasmic reticulum (SR) vesicles. Hence, it is a candidate for use as a substitute for halothane or caffeine in the in vitro contracture test for the diagnosis of susceptibility to malignant hyperthermia (MH). To characterize the effect of DTCAT at cell level, Ca 2+ release experiments were performed on cultured, human skeletal muscle myotubes using the fluorescent Ca 2+ indicator fura2-AM. DTCAT was also assayed in the in vitro contracture test on human skeletal muscle bundles obtained from individuals diagnosed susceptible (MHS), normal (MHN) or equivocal for halothane (MHEH) and compared to the standard test substances caffeine and halothane. DTCAT increased, in a concentration-dependent manner and with a higher efficacy as compared to caffeine, the free, intracellular Ca 2+ levels of cultured MHN and MHS skeletal muscle myotubes. This effect was similar in both types of myotubes and involved the release of Ca 2+ from SR stores as well as Ca 2+ -influx from the extracellular space. Inhibition of ryanodine receptors either with ryanodine or with ruthenium red markedly reduced DTCAT-induced increase in intracellular Ca 2+ concentration while abolishing that induced by caffeine. In MHN skeletal muscle bundles, DTCAT induced contractures with an EC 50 value of 160 ± 91 μM. However, the sensitivity of MHS or MHEH muscles to DTCAT was similar to that of MHN muscles. In conclusion, DTCAT is not suitable for the diagnosis of MH susceptibility due to its failure to discriminate between MHN and MHS muscles.

P-Glycoprotein (Pgp) inhibition by three sets of four isomers of N , N -bis(cyclohexanol)amine aryl esters was assessed on rhodamine 123 (R123) efflux in human MDR1-gene transfected mouse T-lymphoma L5178 cells and on Sf 9 ATPase activity. The most active compounds inhibited Pgp with IC 50 values much lower than those of either cyclosporin A (CSA) or GF120918. As to R123 efflux inhibition, the role of the bond present in the second aryl moiety appeared important since the triple bond derivatives (3a–d) were the most powerful as compared to the double bond (2a–d) and the single bond (1a–d) counterparts. Concentration–inhibition curves of 2c and 3d exhibited a biphasic behaviour suggesting the existence of two binding sites in the recognition domain of Pgp. Persistence of inhibition by these compounds resulted to be intermediate between that caused by CSA and GF120918. R123 exhibited positive interaction with CSA, 1d, 1c, 2d, 2c and 3c, the concentration–inhibition curves being shifted leftward when R123 concentration was increased, while it exhibited negative interaction with 3d and no effect with GF120918. Sf 9 ATPase activity was stimulated in an increasing order of potency by 2c, 3c, 2d, CSA, epirubicin and 3d. In a decreasing order of potency 3d, 2c, GF120918, CSA, 2d and 3c inhibited at sub-nanomolar concentrations epirubicin-stimulated ATPase activity. In conclusion, isomeric geometry and restriction of molecular flexibility of N , N -bis(cyclohexanol)amine aryl esters were crucial for their presentation to and inhibition of Pgp as transport substrates, R123 and epirubicin cooperating with them to this inhibition.