The beneficial health effects of flavonoids on the cardiovascular system: Focus on K+ channels.

Substantial experimental evidences support the hypothesis that dietary flavonoid intake has a favourable impact on cardiovascular diseases such as systemic, arterial hypertension and coronary artery diseases, which represent the leading cause of morbidity and mortality worldwide. The biological effects of flavonoids involve complex biochemical interactions with numerous, specific, cellular and molecular targets. K+ channels, fine modulators of both cardiac action potential and vascular cell membrane potential, represent one of these targets. Overexpression, downregulation or dysfunction of these channel proteins are the cause of many cardiovascular diseases. Therefore, it appears of particular interest a detailed analysis of the flavonoid potential, direct/indirect modulation of cardiovascular K+ channels as these natural compounds ingested with the diet, despite extensive gut metabolism, may accumulate at cellular level in the form of the parent aglycones. The present review will portray their effects on cardiovascular K+ channels. Molecular docking was used to strengthen experimental evidences and describe flavonoid-channel interactions at molecular level.

Fine tuning by protein kinases of CaV1.2 channel current in rat tail artery myocytes.

Seventeen compounds, rather selective, direct or indirect inhibitors and activators of PKA, PKG, and PKC, were analysed for effects on vascular CaV1.2 channel current (ICa1.2) by using the patch-clamp technique in single rat tail artery myocytes. The aim was to investigate how PKs regulate ICa1.2 and disclose any unexpected modulation of CaV1.2 channel function by these agents. The cAMP analogues 8-Br-cAMP and 6-Bnz-cAMP partially reduced ICa1.2 in dialysed cells, while weakly increasing it under the perforated configuration. The ß-adrenoceptor agonist isoproterenol and the adenylate cyclase activator forskolin concentration-dependently increased ICa1.2; this effect was reversed by PKA inhibitors H-89 and KT5720, but not by PKI 6-22. The cGMP analogue 8-Br-cGMP, similarly to the NO-donor SNP, moderately reduced ICa1.2, this effect being reversed to a slight stimulation under the perforated configuration. Among PKG inhibitors, Rp-8-Br-PET-cGMPS decreased current amplitude in a concentration-dependent manner while Rp-8-Br-cGMPS was ineffective. The non-specific phosphodiesterase inhibitor IBMX increased ICa1.2, while H-89, KT5720, and PKI 6-22 antagonized this effect. The PKC activator PMA, but not the diacylglycerol analogue OAG, stimulated ICa1.2 in a concentration-dependent manner; conversely, the PKCα inhibitor Gö6976 markedly reduced basal ICa1.2 and, similarly to the PKCδ (rottlerin) and PKCε translocation inhibitors antagonised PMA-induced current stimulation. The ensemble of findings indicates that the stimulation of cAMP/PKA, in spite of the paradoxical effect of both 8-Br-cAMP and 6-Bnz-cAMP, or PKC pathways enhanced, while that of cGMP/PKG weakly inhibited ICa1.2 in rat tail artery myocytes. Since Rp-8-Br-PET-cGMPS and Gö6976 appeared to block directly CaV1.2 channel, their docking to the channel protein was investigated. Both compounds appeared to bind the α1C subunit in a region involved in CaV1.2 channel inactivation, forming an interaction network comparable to that of CaV1.2 channel blockers. Therefore, caution should accompany the use of these agents as pharmacological tools to elucidate the mechanism of action of drugs on vascular preparations.

Cancer cell permeability-glycoprotein as a target of MDR reverters: possible role of novel dihydropyridine derivatives.

The overexpression of permeability-glycoprotein (P-gp) and other drug transporters (ATP-binding cassette) confers a multidrug resistance (MDR) phenotype on cells in various diseases, including many forms of cancer. Development of MDR is one of the main reasons of failure in malignant tumour chemotherapy, as tumour cells, by increasing drug efflux, acquire cross-resistance to many structurally and functionally unrelated anticancer agents, which therefore never achieve effective intracellular concentrations. Endeavouring to find MDR-reverters is a crucial task for exploring new anti-cancer therapeutic intervention. Although many P-gp inhibitors have so far been identified, it is widely recognised that their interaction with P-gp is a complex process and, presently, the details of the mechanisms of action are still a matter of debate. These compounds turned out, however, to be of limited clinical usefulness owing to their inherent pharmacological activities (first generation compounds) and their accessory, inhibiting activity on CYP enzyme system (second generation compounds). Moreover, recent advances of the knowledge on P-gp structure and function and on the mechanisms of P-gp inhibition will prove fruitful for the development of novel therapeutically effective P-gp inhibitors. A dibenzoyl-1,4-dihydropyridine compound (DP7) has been shown to be a powerful P-gp inhibitor, almost devoid of cardiovascular effects, but capable of inhibiting liver CYP3A. DP7 is considered a lead compound for the development of novel dihydropyridines which do not affect CYP enzyme system but still retain the activity towards ABC-efflux transporters.

(+/-)-Naringenin as large conductance Ca(2+)-activated K+ (BKCa) channel opener in vascular smooth muscle cells.

UNLABELLED: BACKGROUND AND PURPOSE. The aim of this study was to investigate, in vascular smooth muscle cells, the mechanical and electrophysiological effects of (+/-)-naringenin. EXPERIMENTAL APPROACH: Aorta ring preparations and single tail artery myocytes were employed for functional and patch-clamp experiments, respectively. KEY RESULTS: (+/-)-Naringenin induced concentration-dependent relaxation in endothelium-denuded rat aortic rings pre-contracted with either 20 mM KCl or noradrenaline (pIC(50) values of 4.74 and 4.68, respectively). Tetraethylammonium, iberiotoxin, 4-aminopyridine and 60 mM KCl antagonised (+/-)-naringenin-induced vasorelaxation, while glibenclamide did not produce any significant antagonism. Naringin [(+/-)-naringenin 7-beta-neohesperidoside] caused a concentration-dependent relaxation of rings pre-contracted with 20 mM KCl, although its potency and efficacy were significantly lower than those of (+/-)-naringenin. In rat tail artery myocytes, (+/-)-naringenin increased large conductance Ca(2+)-activated K(+) (BK(Ca)) currents in a concentration-dependent manner; this stimulation was iberiotoxin-sensitive and fully reversible upon drug wash-out. (+/-)-Naringenin accelerated the activation kinetics of BK(Ca) current, shifted, by 22 mV, the voltage dependence of the activation curve to more negative potentials, and decreased the slope of activation. (+/-)-Naringenin-induced stimulation of BK(Ca) current was insensitive either to changes in the intracellular Ca(2+) concentration or to the presence, in the pipette solution, of the fast Ca(2+) chelator BAPTA. However, such stimulation was diminished when the K(+) gradient across the membrane was reduced. CONCLUSIONS AND IMPLICATIONS: The vasorelaxant effect of the naturally-occurring flavonoid (+/-)-naringenin on endothelium-denuded vessels was due to the activation of BK(Ca) channels in myocytes.

Functional, electrophysiological and molecular docking analysis of the modulation of Cav 1.2 channels in rat vascular myocytes by murrayafoline A.

BACKGROUND AND PURPOSE: The carbazole alkaloid murrayafoline A (MuA) enhances contractility and the Ca(2+) currents carried by the Cav 1.2 channels [ICa1.2 ] of rat cardiomyocytes. As only few drugs stimulate ICa1.2 , this study was designed to analyse the effects of MuA on vascular Cav 1.2 channels. EXPERIMENTAL APPROACH: Vascular activity was assessed on rat aorta rings mounted in organ baths. Cav 1.2 Ba(2+) current [IBa1.2 ] was recorded in single rat aorta and tail artery myocytes by the patch-clamp technique. Docking at a 3D model of the rat, α1c central pore subunit of the Cav 1.2 channel was simulated in silico. KEY RESULTS: In rat aorta rings MuA, at concentrations ≤14.2 µM, increased 30 mM K(+) -induced tone and shifted the concentration-response curve to K(+) to the left. Conversely, at concentrations >14.2 µM, it relaxed high K(+) depolarized rings and antagonized Bay K 8644-induced contraction. In single myocytes, MuA stimulated IBa1.2 in a concentration-dependent, bell-shaped manner; stimulation was stable, incompletely reversible upon drug washout and accompanied by a leftward shift of the voltage-dependent activation curve. MuA docked at the α1C subunit central pore differently from nifedipine and Bay K 8644, although apparently interacting with the same amino acids of the pocket. Neither Bay K 8644-induced stimulation nor nifedipine-induced block of IBa1.2 was modified by MuA. CONCLUSIONS AND IMPLICATIONS: Murrayafoline A is a naturally occurring vasoactive agent able to modulate Cav 1.2 channels and dock at the α1C subunit central pore in a manner that differed from that of dihydropyridines.

Nitric oxide modulation of interleukin-1[beta]-evoked intracellular Ca2+ release in human astrocytoma U-373 MG cells and brain striatal slices.

Intracellular Ca(2+) mobilization and release into mammal CSF plays a fundamental role in the etiogenesis of fever induced by the proinflammatory cytokine interleukin-1beta (IL-1beta) and other pyrogens. The source and mechanism of IL-1beta-induced intracellular Ca(2+) mobilization was investigated using two experimental models. IL-1beta (10 ng/ml) treatment of rat striatal slices preloaded with (45)Ca(2+) elicited a delayed (30 min) and sustained increase (125-150%) in spontaneous (45)Ca(2+) release that was potentiated by l-arginine (300 microm) and counteracted by N-omega-nitro-l-arginine methyl ester (l-NAME) (1 and 3 mm). The nitric oxide (NO) donors diethylamine/NO complex (sodium salt) (0.3 and 1 mm) and spermine/NO (0.1 and 0.3 mm) mimicked the effect of IL-1beta on Ca(2+) release. IL-1beta stimulated tissue cGMP concentration, and dibutyryl cGMP enhanced Ca(2+) release. The guanyl cyclase inhibitors 1H-[1,2, 4]oxadiazole[4,3-a] quinoxalin-1-one (100 microm) and 6-[phenylamino]-5,8 quinolinedione (50 microm) counteracted Ca(2+) release induced by 2.5 but not 10 ng/ml IL-1beta. Ruthenium red (50 microm) and, to a lesser extent, heparin (3 mg/ml) antagonized IL-1beta-induced Ca(2+) release, and both compounds administered together completely abolished this response. Similar results were obtained in human astrocytoma cells in which IL-1beta elicited a delayed (30 min) increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) (402 +/- 71.2% of baseline), which was abolished by 1 mm l-NAME. These data indicate that the NO/cGMP-signaling pathway is part of the intracellular mechanism transducing IL-1beta-evoked Ca(2+) mobilization in glial and striatal cells and that the ryanodine and the inositol-(1,4,5)-trisphosphate-sensitive Ca(2+) stores are involved.

The effect of pulsed electromagnetic fields on the physiologic behaviour of a human astrocytoma cell line.

We evaluated the effects of 50 Hz pulsed electromagnetic fields (EMFs) with a peak magnetic field of 3 mT on human astrocytoma cells. Our results clearly demonstrate that, after the cells were exposed to EMFs for 24 h, the basal [Ca(2+)](i) levels increased significantly from 124+/-51 nM to 200+/-79 nM. Pretreatment of the cells with 1.2 microM substance P increased the [Ca(2+)](i) to 555+/-278 nM, while EMF exposure caused a significant drop in [Ca(2+)](i) to 327+/-146 nM. The overall effect of EMFs probably depends on the prevailing Ca(2+) conditions of the cells. After exposure, the proliferative responses of both normal and substance P-pretreated cells increased slightly from 1.03 to 1.07 and 1.04 to 1.06, respectively. U-373 MG cells spontaneously released about 10 pg/ml of interleukin-6 which was significantly increased after the addition of substance P. Moreover, immediately after EMF exposure and 24 h thereafter, the interleukin-6 levels were more elevated (about 40%) than in controls. On the whole, our data suggest that, by changing the properties of cell membranes, EMFs can influence Ca(2+) transport processes and hence Ca(2+) homeostasis. The increased levels of interleukin-6 after 24 h of EMF exposure may confirm the complex connection between Ca(2+) levels, substance P and the cytokine network.

Interleukin-1 beta stimulation of 45Ca2+ release from rat striatal slices.

1. Previous observations that centrally injected interleukin-1 beta (IL-1 beta) into rabbits induces a sustained rise in cerebrospinal fluid (CSF) Ca2+ concentration ([Ca2+]) as well as fever, prompted us to undertake an in vitro study to corroborate the in vivo results and gain insight as to the source and mechanism of IL-1 beta-induced Ca2+ mobilization. 2.IL-1 beta treatment of rat striatal slices preloaded with 45Ca2+ elicited a rise in spontaneous 45Ca2+ release which was dose-dependent, delayed in onset and of extended duration. At concentrations of 1, 5 and 10 ng ml-1, the 45Ca2+ efflux increased by 6.3 +/- 1.3 (s.e. mean), 33.4 +/- 5.0 and 159 +/- 10.5% respectively. 3. At 1 microgram ml-1, the specific IL-1 receptor antagonist, IRAP, antagonised the effect induced by, 10 ng ml-1 IL-1. 4. Caffeine 10 mM,which failed to release calcium on its won, potentiated IL-1-elicited 45Ca2+ release. 5. Perfusion with a Ca(2+)-free medium obtained by use of excess EGTA (3 mM) or in the presence of the Ca2+ channel blocker, nifedipine (3 x 10(-8 M) abolished the potentiating effect of caffeine without affecting the IL-1-induced 45Ca2+ release. 6. Preincubation of slices for 4 h with Bordetella pertussis toxin (PTX, 1.3 micrograms ml-1) did not change the pattern of Ca2+ efflux in response to IL-1. 7. In conclusion, these data indicate that IL-1 stimulates calcium release from brain tissue by a specific, receptor-mediated mechanism which partly depends on extracellular calcium but does not involve a PTX-sensitive G protein as part of the transducing signal.

Effects of some sterically hindered phenols on whole-cell Ca(2+) current of guinea-pig gastric fundus smooth muscle cells.

1. The aim of the present study was to investigate the effects of extracellular application of some sterically-hindered phenols, namely 3-t-butyl-4-hydroxyanisole (BHA), 3,5-di-t-butyl-4-hydroxyanisole (DTBHA) and the dimer of BHA, 2,2'-dihydroxy-3,3'-di-t-butyl-5,5'-dimethoxydiphenyl (DIBHA), on the whole-cell Ca(2+) current (I(Ca)) of freshly isolated smooth muscle cells from the guinea-pig gastric fundus, in the presence of a range of Ca(2+) concentrations (1 -- 5 mM) using the patch-clamp technique. The influx of Ca(2+) had characteristics of L-type I(Ca) (I(Ca(L))). 2. BHA as well as DTBHA inhibited I(Ca(L)) in a concentration-dependent manner, during depolarization to 10 mV from a holding potential of -50 mV. Bath application of BHA (50 microM) and DTBHA (30 microM) decreased I(Ca(L)) by 48.9% and 45.2%, respectively. This inhibition was only partially reversible. In contrast, DIBHA (up to 50 microM) was devoided of effects on I(Ca(L)). 3. BHA inhibition of I(Ca(L)) was voltage-dependent and inversely related to the external concentration of Ca(2+). On the other hand, DTBHA inhibition was only voltage-dependent. 4. BHA and DTBHA shifted the voltage range of the steady-state inactivation curve to more negative potentials by 8 mV at the mid-potential of the curve, without affecting the activation curve. Furthermore, BHA and DTBHA did not modify the time-course of the current decay. 5. We conclude that the inhibition of I(Ca(L)) by BHA and DTBHA is qualitatively similar to that of a Ca(2+) channel blocker and is characterized by the stabilizing effect of the inactivated state of the channel.

2,5-Di-t-butyl-1,4-benzohydroquinone (BHQ) inhibits vascular L-type Ca(2+) 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 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.

Increase of extracellular brain calcium involved in interleukin-1 beta-induced pyresis in the rabbit: antagonism by dexamethasone.

1. This study investigates the role of extracellular brain calcium in the hyperthermia induced by interleukin-1 beta (IL-1 beta). 2. Intracerebroventricular (i.c.v.) injection of IL-1 beta (12.5 ng kg-1) in rabbits caused a prompt and sustained rise in cerebrospinal fluid (CSF) Ca2+ concentration ([Ca2+]) followed by enhanced prostaglandin E2 (PGE2) release and hyperthermia. 3. A linear and significant correlation was observed between the increase in [Ca2+] induced by IL-1 beta and the rise in body temperature. 4. Ventriculo-cisternal perfusion with artificial CSF containing the calcium chelator EGTA (1.3 mM) blocked the IL-1-induced PGE2 release and countered the febrile response. 5. I.c.v. administration of dexamethasone (Dex) (2.4 and 24 micrograms kg-1) 100 min prior to IL-1 beta, dose-dependently antagonized the cytokine-induced Ca2+ increase, the PGE2 release and the febrile response. 6. These results suggest that changes in extracellular brain calcium are involved in the regulation of body temperature. In this light, the antipyretic action of Dex may be related to its effect on Ca2+ uptake.

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 Ca(2+)-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 Ca(2+)-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 microM Ca2+ or 500 microM 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 pICso (M) values ranging between 7.4 and 5.7.7. Of the 32 compounds studied only 15 phenolic derivatives exhibited both antispasmogenic andantioxidant activity. Within this subgroup a linear and significant correlation was found betweenantispasmogenic 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.

Oxidative ring-coupling of tyrosine and its derivatives by purified rat intestinal peroxidase.

Intestinal peroxidase was shown to catalyse the oxidative ring-coupling of tyrosine, alpha-methyltyrosine, tyramine and morphine whereas amphetamine was not oxidized to any detectable extent. The oxidative ring-coupling reaction can be monitored by changes in absorbance spectra and the dimers formed in this way with morphine and alpha-methyltyrosine were identified by mass spectrometry. Intestinal peroxidase also catalysed the peroxidatic oxidation of L-DOPA and alpha-methyl-L-DOPA, but in this case the reaction would be expected to be more complicated and to yield a variety of possible products. The kinetic parameters for the oxidation of each of these substrates were determined. Since the products of the oxidative ring-coupling reactions may have different pharmacological properties to those of the parent compounds, these studies suggest that, in the presence of an adequate supply of metabolically produced hydrogen peroxide, the action of intestinal peroxidase may affect the behaviour and pharmacokinetics of these compounds after oral administration.

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.

Evidence of a coupled mechanism between monoamine oxidase and peroxidase in the metabolism of tyramine by rat intestinal mitochondria.

The relationship between monoamine oxidase (EC 1.4.3.4; MAO) and peroxidase (EC 1.11.1.7; POD) in the metabolism of tyramine was investigated using the crude mitochondrial fraction of rat intestine. When tyramine was incubated with mitochondria, the formation of the peroxidase-catalysed oxidation product, 2,2'-dihydroxy-5,5'-bis(ethylamino)diphenyl (dityramine), identified by mass spectrometric analysis, was monitored spectrophotometrically. After an initial lag time, the formation rate of dityramine was linear up to 2 hr, amounting to 17 nmol x hr(-1) x mg protein(-1). A similar value was found for the oxidative deamination of tyramine catalysed by intestinal MAO. Either 10(-3) M clorgyline or 10(-3) M NaCN suppressed this reaction by completely inhibiting MAO or POD, respectively. In the former case, however, addition of H2O2 to the incubation mixture promptly started the reaction. Selective inhibition of MAO-A and MAO-B was achieved with 3 x 10(-7) M clorgyline and 3 x 10(-7) M deprenyl, respectively, and the formation rate of dityramine decreased in a corresponding manner. Preincubation with histamine or spermidine reduced the lag time without affecting the steady-state reaction rate. Higher levels of dityramine were also detected in vivo in rat intestine after oral administration of tyramine. These results indicate that the peroxidase-dependent metabolism of tyramine in the gut may be driven by H2O2 produced by MAO activities and that MAO-A is mainly responsible for this process, as well as for the oxidative deamination of tyramine.

3,5-di-t-butyl-4-hydroxyanisole (DTBHA) activation of rat skeletal muscle sarcoplasmic reticulum Ca(2+)-ATPase.

3,5-Di-t-butyl-4-hydroxyanisole (DTBHA) increased in a concentration-dependent manner (calculated pEC(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.

Peroxidase catalysed formation of prostaglandins from arachidonic acid.

Horseradish peroxidase and bovine lactoperoxidase (EC 1.11.1.7), when incubated aerobically with arachidonate, gave rise to the formation of substances identified by bioassay as prostaglandin F2 alpha (PGF2 alpha)- and prostaglandin E2 (PGE2)-like compounds. Boiling of enzymes, which suppressed their capacity to peroxidize guaiacol, also destroyed their capacity to convert arachidonate into PG-like compounds. The rates of formation of PG-like compounds rapidly declined with time, approaching zero after 10 and 20 min for PGE2 alpha- and PGE2-like compounds, respectively. Addition of more enzyme further promoted the reaction. Horseradish and lacto-peroxidases showed optimum pH values of 9.0 and 10.0, respectively. Both enzymes exhibited apparent Km values of about 5 x 10(-5) M for arachidonate. Some reducing agents such as ascorbic acid, NADH and adrenaline dose-dependently inhibited this reaction. The haem poison, phenylhydrazine, also inhibited, with an IC50 of 1 x 10(-7) M. Indomethacin inhibited only the formation of PGE2-like compounds with an IC50 of about 3 x 10(-6) M. As compared to a standard commercial preparation of horseradish peroxidase, the purified horseradish basic and acidic isoenzymes exhibited a higher activity, towards arachidonate whereas other haemoproteins, possessing peroxidase activity, were less active. TLC and GC-MS analyses performed on the reaction products led to the identification of PGF2 alpha, PGE2 and PG6K1 alpha and other unidentified arachidonate derivatives. At 25 degrees, pH 9.5, horseradish peroxidase, acting on saturating concentration of arachidonate, catalysed the formation of 60 mumol/min/mmole enzyme of PGE2 + PGF2 alpha. This appears to be the first report of the synthesis of prostaglandins catalysed by peroxidases.

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.

Effect of loxiglumide (CR 1505) on CCK-induced contractions and 3H-acetylcholine release from guinea-pig gallbladder.

Release of [3H]-acetylcholine (3H-ACh) and muscle contractions in response to cholecystokinin (CCK) were measured and recorded simultaneously from isolated guinea-pig gallbladder. Cholecystokinin octapeptide (CCK8) (10(-10)-10(-7) M) enhanced the release of [3H]ACh and the contractions of the muscle. TTX (10(-6) M) inhibited the CCK-induced release of 3H-ACh by only 30%. In Ca(2+)-free medium CCK8 had no effect. Loxiglumide, (CR 1505), a newly synthesized nonpeptide CCK-A-receptor antagonist, D.L-(3,4-dichlorbenzoilamino)-5-/N-(3-methoxypropyl)-pentylamin o-5-oxo-pentanoi c acid, antagonized both the ACh-releasing effect of CCK and the contractions in a dose-dependent manner. The affinity (pA2) of CR 1505 to CCK-receptors, determined by the shift of the concentration-response curves for CCK8 was 8.36. It was 5 logarithmic orders higher than the pA2 of proglumide. The IC50 value of CR 1505 calculated by the CCK-induced release of 3H-ACh was 10 nM. The results suggest the existence not only of muscular CCK receptors but also neuronal receptors for CCK probably located on cholinergic nerves.

The mechanism of actions of collagenase on the inhibition of release of acetylcholine from synaptosomal preparations.

The release of acetylcholine from synaptosomal preparations from bovine superior cervical ganglia and rat cortex was inhibited when the preparations were pretreated with collagenase. The inhibition of release could be overcome with the calcium ionophore A23187. Collagenase treatment was shown to inhibit the uptake of calcium into the preparations. In addition, gel electrophoresis of synaptosomal membranes revealed two missing high molecular weight proteins when either synaptosomes or synaptosomal membranes were incubated with collagenase.