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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

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.

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.

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.

In skeletal muscle sarcoplasmic reticulum vesicles, 3,5-di-t-butyl catechol (DTCAT) promotes the release of Ca(2+) through the activation of ryanodine receptor Ca(2+) channels. DTCAT mechanical and electrophysiological effects have now been investigated in rat aorta rings and single tail artery myocytes. Rat aorta rings incubated with either 30 microM ryanodine or 100 microM DTCAT developed tension, which averaged 36% and 7%, respectively, of that induced by phenylephrine. DTCAT reduced concentration-dependently both aorta ring contractions to high K(+) (IC(50)=13.5 microM) and L-type Ba(2+) current (IC(50)=22.0 microM) in isolated myocytes. Tetraethylammonium did not reverse the antispasmodic effect of DTCAT in rings stimulated with either 25 or 60 mM K(+). DTCAT relaxed concentration-dependently phenylephrine-pre-contracted rings with intact endothelium (IC(50)=10.9 microM). This effect was markedly reduced by pre-incubation of rings with 100 microM Nomega-nitro-l-arginine methyl ester. DTCAT antagonised phenylephrine-induced contractions in endothelium-deprived rings, either in the presence or in the absence of ryanodine (IC(50)=18.7 microM and 39.8 microM, respectively). Furthermore, both DTCAT (IC(50)=53.3 microM) and ryanodine reduced significantly the response to phenylephrine in the absence of extracellular Ca(2+). Phenylephrine-stimulated influx of extracellular Ca(2+) was markedly inhibited when tissues were pre-treated with DTCAT (IC(50)=19.0 microM) as well as nifedipine. DTCAT (>100 microM) was also able to antagonise the contractions induced by phorbol 12-myristate, 13-acetate. In conclusion, this is the first demonstration that DTCAT inhibits vascular smooth muscle voltage-operated Ca(2+) channels and promotes the release of endothelial nitric oxide. Ryanodine receptor Ca(2+) channels activation or the impairment of the contractile apparatus by DTCAT seem to play a secondary role in its vascular activity.

Vasoactive intestinal peptide (VIP) was tested for its capability to protect the intrinsic nerves of guinea-pig urinary bladder from damage due to anoxia/glucopenia and reperfusion. Guinea-pig detrusor strips were mounted for tension recording in small organ baths and the nerves were subjected to electric field stimulation. VIP (0.3 microM) improved significantly the response of strips to electrical field stimulation either during anoxia/glucopenia or thereafter during reperfusion, as compared to untreated tissues. The antioxidant activity of VIP assessed as its capability to scavenge peroxyl radicals during linoleic acid oxidation corresponded to 6.42+/-0.13 pIC(50) M, i.e. close to the concentration proved to protect strips against the anoxic--glucopenic and reperfusion damage.

The antispasmodic effects of 3- t -butyl-4-hydroxyanisole (BHA) and some structurally related compounds were investigated in endothelium-intact rat aorta rings. Nordihydroguaieretic acid (NDGA), BHA, 3,5-di- t -butyl-4-hydroxyanisole (DTBHA), 2,6-di-isopropyl phenol (propofol) and 2,2′-dihydroxy-3,3′-di- t -butyl-5,5′-dimethoxydiphenyl (DIBHA) did not cause relaxation when added at the plateau of phenylephrine-evoked contraction, nor did they affect the concentration–relaxation curve for acetylcholine in precontracted rings. In rings depolarised with physiological salt solution (PSS) containing 40 mM K + , NDGA, BHA, DTBHA, 2,5-di- t -butyl-1,4-benzohydroquinone (BHQ), propofol and nifedipine, but not DIBHA, inhibited the contraction induced by cumulative addition of Ca 2+ (0.05–10 mM) in a concentration-dependent manner; this inhibition was inversely related to the Ca 2+ concentration. In 40 mM K + PSS, 25 nM nifedipine blocked the 1 mM Ca 2+ -induced contraction, whereas 50 μM DTBHA, NDGA, BHA, BHQ and propofol significantly antagonised it by 84.4%, 73.0%, 52.8%, 45.6% and 35.7%, respectively. In the presence of 1 μM methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyridine-5-carboxylate (Bay K 8644), the response to Ca 2+ did not differ from control values with nifedipine and BHQ, was partially restored with DTBHA and NDGA, and was not affected with BHA and propofol. Nifedipine markedly inhibited (85.2%) the Ba 2+ -induced contraction and this effect was totally reversed by Bay K 8644. BHA and DTBHA showed antispasmodic activity (45.3% and 43.1%, respectively) which was partly reversed by Bay K 8644. In contrast, Bay K 8644 did not affect the inhibition exerted by BHQ, NDGA and propofol (69.5%, 53.3% and 46.1%, respectively). Nifedipine, BHA, DTBHA, propofol and NDGA inhibited the contractile response to 1 mM Ca 2+ of aorta rings depolarised with 40 or 80 mM K + PSS to a similar extent. Cromakalim inhibited the Ca 2+ -evoked contraction only in 30 mM K + PSS and BHQ only in 80 mM K + PSS. DIBHA had no effect on this model. Cromakalim, but not BHA, stimulated 86 Rb + efflux from ring preparations. In 80 mM K + PSS containing 1 μM nifedipine, only papaverine affected the phenylephrine-induced contraction. Moreover, when the rings were preincubated with 1 mM Ni 2+ , the response to phenylephrine in the presence of BHQ was significantly reduced. In conclusion, we propose that BHA may non-specifically inhibit Ca 2+ influx at the plasmalemma level rather than affect the function of K + channels, Ca 2+ release from intracellular stores or endothelium-dependent relaxation.