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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.

This study investigates the mechanism whereby the antioxidant 2-t-butyl-4-methoxyphenol (BHA) relaxes guinea pig gastric fundus smooth muscle. In circular smooth muscle strips, 10 microM cyclopiazonic acid, a specific inhibitor of sarcoplasmic reticulum Ca2+-ATPase, induced a prolonged rise in tension which depended on the presence of extracellular Ca2+. BHA (pIC50 = 5.83), sodium nitroprusside (6.85), isoproterenol (7.69) and nifedipine (8.02), but not 2,6-di-t-butyl-4-methoxyphenol (DTBHA) (up to 30 microM), relaxed muscle strips contracted with cyclopiazonic acid. Methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyri dine-5-carboxylate (Bay K 8644) (1 microM) antagonised the nifedipine- but not the BHA-induced relaxation. Nifedipine and isoproterenol (10 microM) caused a decrease in spontaneous tone, but did not counteract the subsequent rise in tension elicited by 10 microM cyclopiazonic acid. Conversely, 100 microM BHA and 100 microM sodium nitroprusside not only significantly reduced spontaneous tone but also markedly impaired the response of the muscles to cyclopiazonic acid. DTBHA failed to show either effect. When added to preparations completely relaxed by 100 microM BHA, 10 mM tetraethylammonium still elicited nifedipine-sensitive tonic and phasic contractions in the presence or absence of 10 microM cyclopiazonic acid. BHA and DTBHA inhibited, in a concentration-dependent manner, the Ca2+-promoted contraction of strips depolarised by 10 mM tetraethylammonium. The BHA antagonism showed a non-competitive profile while that of DTBHA was competitive. In muscle strips at rest, 10 microM BHA caused a significant increase in tissue cAMP concentration, leaving cGMP unmodified. To conclude, the myorelaxant action of BHA on gastric fundus smooth muscle appears to be mediated partly by an increase in cAMP levels and partly by inhibition of Ca2+ influx from the extracellular space.

To characterise the pharmacological activity of 2,5-di- t -butyl-1,4-benzohydroquinone (BHQ) on vascular smooth muscle, the different effects of BHQ on rat aorta were investigated under several experimental conditions. In aortic rings at rest or depolarised with 80 mM K + in the presence of 1 μ M nifedipine, BHQ evoked a slow tonic contraction which was antagonised by 1 mM Ni 2+ . Depolarised rings contracted in response to addition of 1 mM Ca 2+ , with an EC 50 value of 32.4±1.0 mM for K + . At 20 mM K + , Ca 2+ -induced contraction was enhanced by BHQ. This effect was antagonised by 1 mM Ni 2+ , but not by 1 μ M nifedipine. By contrast, at 40, 80 and 128 mM K + , BHQ antagonised Ca 2+ -induced contraction. This effect was partially reversed by 1 μ M methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyridine-5-carboxylate (Bay K 8644) or by increasing extracellular Ca 2+ concentration. In the presence of nifedipine and Ni 2+ , depolarised rings (80 mM K + ) contracted in response to addition of 1 μ M phenylephrine; this response was fast and then slowly decreased. When the preparations were preincubated with BHQ, the phenylephrine-induced contraction was transient and antagonised in a concentration-dependent manner by BHQ. These results indicate that the myotonic effect of BHQ on rat aortic rings depends on activation of Ca 2+ influx via a Ni 2+ -sensitive pathway, whereas its myolytic activity is due either to antagonism of Ca 2+ entry via L-type Ca 2+ channels or depletion of intracellular Ca 2+ stores.

Taurine neuroinhibitory features have suggested its potential for neuroprotection. The aim of the present study was to assess whether it prevents or counteracts brain ischemia and reperfusion-induced cell injury. Rat brain cortical slices were subjected to oxygen/glucose deprivation and reperfusion. Tissue damage was assessed by measuring the release of glutamate and lactate dehydrogenase (LDH) during reperfusion and by determining final tissue water gain, taken as an index of cell swelling. When added during the reperfusion period taurine did not significantly affect oxygen/glucose deprivation-induced LDH and glutamate release, while it antagonised tissue water gain in a concentration-dependent manner (IC 50 = 46.5 µM). The latter effect was antagonised by 50% when a taurine transport inhibitor, 2-(guanidino)ethanesulphonic acid (GES), or a GABA A receptor antagonist, bicuculline, was added together with taurine, while it was completely abolished when both GES and bicuculline or the volume-sensitive outwardly rectifying (VSOR) Cl − channel blocker, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), was used. On the contrary, when present throughout the entire experiment, taurine significantly reduced oxygen/glucose deprivation-induced LDH and glutamate release with a maximal effect (45% reduction) between 5 and 20 mM. Taurine antagonised also tissue water gain according to a “U-shaped” concentration–response curve, which was significant within the range of 0.01–1.0 mM concentration. This effect was partially counteracted by GES as well as by bicuculline and fully reverted by NPPB. In conclusion, since brain edema is a major contributing factor to morbidity and mortality in stroke, the present findings give the rational basis for assessing taurine efficacy in reducing brain edema in vivo .

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.

The aim of this work was to clarify the mechanism by which 2,5-di- t -butyl-1,4-benzohydroquinone (BHQ) induces relaxation of rat thoracic aorta. In particular, the role of endothelium-derived nitric oxide (NO) was investigated. BHQ concentration dependently (0.1–10 μM) relaxed rat aorta rings precontracted with phenylephrine. This effect was dependent on the intactness of the endothelium, suppressed by preincubation with 100 μM N ω-nitro- l -arginine methyl ester and antagonised by 3–30 μM methylene blue. The 10 μM BHQ-induced relaxation, however, was followed by the gradual and slow return to phenylephrine-induced tone. Superoxide dismutase (250 U/ml) increased the BHQ-induced relaxation, while preincubation with 3 mM diethyldithiocarbamate inhibited it in a time-dependent fashion. BHQ gave rise to superoxide anion formation which was markedly inhibited by the addition of superoxide dismutase (250 U/ml), either in the presence or in the absence of aorta rings. The non-specific blocker of Ca 2+ channels, Ni 2+ , concentration dependently attenuated the BHQ relaxing effect. BHQ did not modify the relaxation induced by the NO donor 3-morpholino-sydnonimine in endothelium-deprived rings. In conclusion, BHQ induces endothelium-dependent relaxation and gives rise, by auto-oxidation, to the formation of superoxide anion. The former effect results from the enhanced synthesis of NO rather than from its enhanced biological activity; NO synthase is presumed to be stimulated by BHQ-induced activation of Ca 2+ influx through Ni 2+ -sensitive Ca 2+ channels.