Fabio Fusi

Potentiation of mitochondrial Ca2+ sequestration by taurine

The effects of taurine (2-aminoethanesulphonic acid) and its analogues, 2-aminoethylarsonic acid, 2-hydroxyethanesulphonic (isethionic) acid, 3-aminopropanesulphonic acid, 2-aminoethylphosphonic acid, and N,N-dimethyltaurine, were studied on the transport of Ca2+ by mitochondria isolated from rat liver. Taurine enhanced Ca2+ uptake in an apparently saturable process, with a Km value of about 2.63 mM. Taurine behaved as an uncompetitive activator of Ca2+ uptake, increasing both the apparent Km and Vmax values of the process. This effect was not modified in the presence of cyclosporin A (CsA). N,N-Dimethyltaurine also stimulated Ca2+ uptake at higher concentrations, but there was no evidence that the process was saturable over the concentration range used (1-10 mM). Aminoethylarsonate was a weak inhibitor of basal Ca2+ uptake, but inhibited that stimulated by taurine in an apparently competitive fashion (Ki = 0.05 mM). The other analogues had no significant effects on this process. Taurine either in the presence or the absence of CsA had no effect on Ca2+ release induced by 200 nM ruthenium red. Thus, the mechanism of taurine-enhanced Ca2+ accumulation appears to involve stimulation of Ca2+ uptake via the uniport system rather than inhibition of Ca2+ release via the ion (Na+/Ca2+ and/or H+/Ca2+) exchangers or by taurine modulating the permeability transition of the mitochondrial inner membrane. Overall, these findings indicate an interaction of taurine with an as yet unidentified mitochondrial site which might regulate the activity of the uniporter. The unique role of taurine in modulating mitochondrial Ca2+ homeostasis might be of particular importance under pathological conditions that are characterised by cell Ca2+ overload, such as ischaemia and oxidative stress.

Calcium antagonist and antiperoxidant properties of some hindered phenols

1 The calcium antagonist and antioxidant activities of certain synthetic and natural phenols, related to BHA (2‐t‐butyl‐4‐methoxyphenol), were evaluated in rat ileal longitudinal muscle and in lipid peroxidation models respectively. 2 Compounds with a phenol or a phenol derivative moiety, with the exception of 2,2′‐dihydroxy‐3,‐3′‐di‐t‐butyl‐5,5′‐dimethoxydiphenyl (di‐BHA), inhibited in a concentration‐dependent manner the BaCl2‐induced contraction of muscle incubated in a Ca2+‐free medium. Calculated pIC50 (m) values ranged between 3.32 (probucol) and 4.96 [3,5‐di‐t‐butyl‐4‐hydroxyanisole (di‐t‐BHA)], with intermediate activity shown by khellin < gossypol < quercetin < 3‐t‐butylanisole < BHA < nordihydroguaiaretic acid (NDGA) < 2,6‐di‐t‐butyl‐4‐methylphenol (BHT) and papaverine. 3 The Ca2+ channel activator Bay K 8644 overcame the inhibition sustained by nifedipine, BHA and BHT, while only partially reversing that of papaverine. 4 BHA, BHT, nifedipine and papaverine also inhibited in a concentration‐dependent fashion CaCl2 contractions of muscle depolarized by a K+‐rich medium. This inhibition appeared to be inversely affected by the Ca2+‐concentration used. 5 The inhibitory effects of nifedipine, papaverine, BHA and BHT were no longer present when muscle contraction was elicited in skinned fibres by 5 μm Ca2+ or 500 μm Ba2+, suggesting a plasmalemmal involvement of target sites in spasmolysis. 6 Comparative antioxidant capability was assessed in two peroxyl radical scavenging assay systems. These were based either on the oxidation of linoleic acid initiated by a heat labile azo compound or on lipid peroxidation of rat liver microsomes promoted by Fe2+ ions. Across both model systems, di‐t‐BHA, NDGA, BHT, di‐BHA, BHA and quercetin ranked as the most potent inhibitors of lipid oxidation, with calculated pIC50 (m) values ranging between 7.4 and 5.7. 7 Of the 32 compounds studied only 15 phenolic derivatives exhibited both antispasmogenic and antioxidant activity. Within this subgroup a linear and significant correlation was found between antispasmogenic activity and antioxidation. These bifunctional compounds were characterized by the presence of at least one hydroxyl group on the aromatic ring and a highly lipophilic area in the molecule. 8 Di‐t‐BHA is proposed as a lead reference compound for future synthesis of new antioxidants combining two potentially useful properties in the prevention of tissue damage after ischaemia‐reperfusion injury.

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

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

Quercetin as a novel activator of L‐type Ca2+ channels in rat tail artery smooth muscle cells

The aim of this study was to investigate the effects of quercetin, a natural polyphenolic flavonoid, on voltage‐dependent Ca2+ channels of smooth muscle cells freshly isolated from the rat tail artery, using either the conventional or the amphotericin B‐perforated whole‐cell patch‐clamp method. Quercetin increased L‐type Ca2+ current [ICa(L)] in a concentration‐ (pEC50=5.09±0.05) and voltage‐dependent manner and shifted the maximum of the current‐voltage relationship by 10 mV in the hyperpolarizing direction, without, however, modifying the threshold and the equilibrium potential for Ca2+. Quercetin‐induced ICa(L) stimulation was reversible upon wash‐out. T‐type Ca2+ current was not affected by quercetin. Quercetin shifted the voltage dependence of the steady‐state inactivation and activation curves to more negative potentials by about 5.5 and 7.5 mV respectively, in the mid‐potential of the curves as well as increasing the slope of activation. Quercetin slowed both the activation and the deactivation kinetics of the ICa(L). The inactivation time course was also slowed but only at voltages higher than 10 mV. Moreover quercetin slowed the rate of recovery from inactivation. These results prove quercetin to be a naturally‐occurring L‐type Ca2+ channel activator.

2,5-Di-t-butyl-1,4-benzohydroquinone (BHQ) inhibits vascular L-type Ca2+ channel via superoxide anion generation

The aim of the present study was to investigate the effects of 2,5‐di‐t‐butyl‐1,4‐benzohydroquinone (BHQ), an inhibitor of the sarco‐endoplasmic reticulum Ca2+‐ATPase (SERCA), on the whole‐cell voltage‐dependent L‐type Ca2+ current (ICa(L)) of freshly isolated smooth muscle cells from the rat tail artery using the patch‐clamp technique. BHQ, added to the perfusion solution, reduced ICa(L) in a concentration‐ (IC50=66.7 μM) and voltage‐dependent manner. This inhibition was only partially reversible. BHQ shifted the voltage dependence of the steady‐state inactivation curve to more negative potentials by 7 mV in the mid‐potential of the curve, without affecting the activation curve as well as the time course of ICa(L) inactivation. Preincubation of the cells either with 10 μM cyclopiazonic acid, a SERCA inhibitor, or with 3 mM diethyldithiocarbamate, an inhibitor of intracellular superoxide dismutase (SOD), did not modify BHQ inhibition of ICa(L). On the contrary, this effect was no longer evident when SOD (250 u ml−1) was added to the perfusion medium. Either in the presence or in the absence of cells, BHQ gave rise to superoxide anion formation, which was markedly inhibited by the addition of SOD. These results indicate that, at micromolar concentrations, BHQ inhibits vascular ICa(L) by giving rise to the formation of superoxide anion which in turn impairs the channel function.

Rectal temperature and prostaglandin E2 increase in cerebrospinal fluid of conscious rabbits after intracerebroventricular injection of hemoglobin

Abstract Fever accompanies subarachnoid hemorrhage (SAH) in the majority of patients. In a previous study, hemoglobin (Hb) was shown to catalyze in vitro, under aerobic conditions, the conversion of arachidonic acid to prostaglandin E2 (PGE2) and prostaglandin F2α. The aim of the present work was to assess whether this pathway also operates in vivo and to provide a mechanism to explain post-SAH fever. To this end, PGE2 concentration was determined in cerebrospinal fluid (CSF) of conscious rabbits chronically cannulated in the lateral ventricle and cisterna magna, following intracerebroventricular (i.c.v.) injection of 10 µg or 100 µg of commercial rabbit bicrystallized Hb as a model of SAH. Before i.c.v. injection, Hb solutions were filtered on a polimixin-B column to remove substantially, by over 90%, endotoxin-like substances. Results show that in nine rabbits injection of 10 µg Hb did not significantly modify body temperature or significantly alter CSF PGE2 content. On the contrary, in nine rabbits, injection of 100 µg Hb produced a significant increase in core temperature which was accompanied by a significant increase in CSF PGE2. When data related to these two parameters from the 9 control and 18 Hb-treated rabbits were analyzed as a single group, a linear, positive, and highly significant correlation was found. These findings indicate that, once Hb is released into the subarachnoid space during SAH, it enhances CSF PGE2 content and elicits hyperthermia, thus offering an explanation for the fever that is an aggravating condition in most SAH patients.

Interaction of butylated hydroxyanisole with mitochondrial oxidative phosphorylation.

The antioxidant, butylated hydroxyanisole (BHA), has a number of effects on mitochondrial oxidative phosphorylation. In this study we apply the novel approach developed by Brand (Brand MD, Biochim Biophys Acta 1018: 128-133, 1990) to investigate the site of action of BHA on oxidative phosphorylation in rat liver mitochondria. Using this approach we show that BHA increases the proton leak through the mitochondrial inner membrane and that it also inhibits the delta p (proton motive force across the mitochondrial inner membrane) generating system, but has no effect on the phosphorylation system. This demonstrates that compounds having pleiotypic effects on mitochondrial oxidative phosphorylation in vitro can be analysed and their many effects distinguished. This approach is of general use in analysing many other compounds of pharmacological interest which interact with mitochondria. The implications of these results for the mechanism of interaction of BHA with mitochondrial oxidative phosphorylation are discussed.

3,5‐Dibenzoyl‐4‐(3‐phenoxyphenyl)‐1,4‐dihydro‐2,6‐dimethylpyridine (DP7) as a new multidrug resistance reverting agent devoid of effects on vascular smooth muscle contractility

The aim of this study was to investigate the effects of 3,5‐diacetyl‐ (DP1–DP5) and 3,5‐dibenzoyl‐1,4‐dihydropyridines (DP6–DP11) on vascular functions in vitro, by comparing their mechanical and electrophysiological actions in rat aorta rings and single rat tail artery myocytes, respectively, and to quantify their multidrug resistance (MDR)‐reversing activity in L5178 Y mouse T‐lymphoma cells transfected with MDR1 gene. In rat aorta, the 11 compounds tested, but 3,5‐dibenzoyl‐4‐(3‐phenoxyphenyl)‐1,4‐dihydro‐2,6‐dimethylpyridine (DP7), 3,5‐dibenzoyl‐4‐(3‐chlorophenyl)‐1,4‐dihydro‐2,6‐dimethylpyridine (DP9), 3,5‐dibenzoyl‐4‐(4‐chlorophenyl)‐1,4‐dihydro‐2,6‐dimethylpyridine (DP10) and 3,5‐dibenzoyl‐4‐phenyl‐1,4‐dihydro‐2,6‐dimethylpyridine (DP11), antagonized 60 mM K+ (K60)‐induced contraction in a concentration‐dependent manner, with IC50 (M) values ranging between 5.65 × 10−7 and 2.23 × 10−5. The 11 dihydropyridines tested, but DP7, inhibited L‐type Ca2+ current recorded in artery myocytes in a concentration‐dependent manner, with IC50 (M) values ranging between 1.12 × 10−6 and 6.90 × 10−5. The K+‐channel opener cromakalim inhibited the Ca2+‐induced contraction in K30 but not that evoked in K60. On the contrary, DP7 was ineffective in both experimental conditions. When the rings were preincubated with 1 mM Ni2+ plus 1 μM nifedipine, the response to phenylephrine was significantly reduced by 2,5‐di‐t‐butyl‐1,4‐benzohydroquinone (BHQ), a well‐known endoplasmic reticulum Ca2+‐ATPase inhibitor. DP7 had no effects on this model system. In L5178 MDR cell line, the 11 dihydropyridines tested, but 3,5‐diacetyl‐4‐(2‐nitrophenyl)‐1,4‐dihydro‐2,6‐dimethylpyridine (DP1), 3,5‐diacetyl‐4‐(3‐phenoxyphenyl)‐1,4‐dihydro‐2,6‐dimethylpyridine (DP2) and 3,5‐diacetyl‐4‐(3‐chlorophenyl)‐1,4‐dihydro‐2,6‐dimethylpyridine (DP4), exhibited an MDR‐reversing activity, with IC50 values ranging between 3.02 × 10−7 and 4.27 × 10−5, DP7 being the most potent. In conclusion, DP7 may represent a lead compound for the development of potent dihydropyridine MDR chemosensitizers devoid of vascular effects.

Mechanism of osthole inhibition of vascular Cav1.2 current

Osthole is a coumarin extracted from Cnidium monnieri (L.) Cusson. The medicinal plant is widely used in Vietnamese as well as Chinese traditional medicine as a vasodilating and antihypertensive agent. Here we have tested the proposition that the block of Ca(v)1.2 channels is mainly responsible for its vascular activity. An in-depth analysis of the effect of osthole on Ca(v)1.2 current (I(Ca1.2)) was performed in rat tail artery myocytes using the whole-cell patch-clamp method. Osthole decreased I(Ca1.2) in a concentration- and voltage-dependent manner. At holding potentials of -50 and -80mV, the pIC(50) values were 4.78±0.07 and 4.36±0.08, respectively; the latter corresponded to the drug apparent dissociation constant for resting channels, K(R), of 47.8μM. Osthole speeded up the inactivation kinetics of I(Ca1.2) and shifted the voltage dependence of the inactivation curve to more negative potentials in a concentration-dependent manner, with an apparent dissociation constant for inactivated channels (K(I)) of 6.88μM. Block of I(Ca1.2) was frequency-dependent and the rate of recovery from inactivation was slowed down. In conclusion, osthole is a vascular Ca(v)1.2 channel antagonist stabilizing the channel in its inactivated state. This mechanism may account for the systolic blood pressure reduction induced by the drug in animal models of hypertension and points to osthole as a lead for the development of novel antihypertensive agents.

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

BACKGROUND AND PURPOSE Previous studies have pointed to the plant flavonoids myricetin and quercetin as two structurally related stimulators of vascular Cav1.2 channel current (ICa1.2). Here we have tested the proposition that the flavonoid structure confers the ability to modulate Cav1.2 channels.