Barbara Burghardt

Antiperoxidant effects of dihydropyridine calcium antagonists.

Etude du potentiel antiperoxydant de 5 antagonistes du Ca 2+ dans des liposomes prepares a partir de phospholipides issus de myocarde de rat: Niludipine, Ninodipine, Nisoldipine, Nicardipine et Telodipine. On montre que ces antagonistes du Ca 2+ ont une large capacite antioxydante et peuvent proteger les phospholipides de la membrane du myocarde contre des agents peroxydants

Protection of cardiac membrane phospholipid against oxidative injury by calcium antagonists

Calcium antagonists representative of the four major chemical classes were assessed for their abilities to prevent peroxidation of rat heart membrane lipids through xanthine oxidase-dependent, superoxide-driven, iron-promoted oxygen radical chemistry. The dihydropyridines nifedipine and nitrendipine did not affect peroxidation, even at a concentration (500 microM) approaching their solubility limit. The benzothiazepine diltiazem did protect the cardiac lipids against oxidative injury, but at high micromolar concentrations: 50% inhibition of peroxidation (antiperoxidant IC50) required 510 microM diltiazem. The phenylalkylamines verapamil and gallopamil (D-600) were likewise weak antiperoxidants (approximately 35% inhibition of peroxidation at 500 microM). In contrast, two other alkylamines, bepridil and prenylamine, were very effective membrane lipid protectants with respective antiperoxidant IC50 values of 55 and 75 microM. The diphenylpiperazines flunarizine (IC50 = 190 microM) and cinnarizine (IC50 = 180 microM) displayed moderate antiperoxidant activity. No Ca2+ antagonist inhibited xanthine oxidase under conditions whereby 10 microM allopurinol inhibited enzyme activity by 50%. The effects of the Ca2+ antagonist-antiperoxidants on the kinetics of cardiac membrane lipid peroxidation indicate that they inhibit peroxidation by intercepting oxy- and/or lipid free radical intermediates. These data raise the possibility that antiperoxidant action may contribute to the spectrum of pharmacologic and therapeutic activities of certain Ca2+ antagonists.

Analysis of cardiac membrane phospholipid peroxidation kinetics as malondialdehyde: nonspecificity of thiobarbituric acid-reactivity.

When exposed to xanthine oxidase (superoxide)-dependent, iron-promoted Fenton chemistry, purified cardiac membranes evidenced, by the thiobarbituric acid (TBA) test, a virtually instantaneous peroxidative response with a maximal linear rate of 5.8 nmol malondialdehyde (MDA)-equivalents/mEquivalents lipid ester reacted/min. Yet when the lipids purified from these same membranes and reconstituted into liposomes were peroxidized under identical reaction conditions, the TBA test indicated that a pronounced (∼20-min) lag period preceded a maximal peroxidation rate of only 2.1 nmol MDA-equivalents/ mEquivalents lipid ester reacted/min. After 120 min of peroxidation, the cardiac membranes yielded some 300 nmol TBA-reactive MDA-equivalents/mEquivalent ester, whereas the isolated membrane lipids evidenced ∼40% less TBA-reactivity. To verify that these quantitative and kinetic differences in membrane (phospho)-lipid peroxidation occurred with removal of the lipids from their membrane milieu, the MDA produced during both cardiac membrane peroxidation and the peroxidation of the lipids derived therefrom was isolated as its free anion by ion-pair high-pressure liquid chromatography. As quantified spectrophotometrically, true MDA production during myocardial membrane peroxidation was identical in kinetics and in amount to the production of TBA-reactive substance from the peroxidized isolated membrane lipids. These results demonstrate that significant non-MDA. TBA-reactive species are generated during the peroxidation of cardiac membranes, especially before the maximal rates of bona fide MDA production. As a direct consequence, artifactual levels and kinetics of membrane lipid peroxidation do result.

Thiobarbituric acid-reactive malondialdehyde formation during superoxide-dependent, iron-catalyzed lipid peroxidation: Influence of peroxidation conditions

A systematic study of the influence of biological lipid peroxidation conditions on lipid hydroperoxide decomposition to thiobarbituric acid-reactive malondialdehyde is presented. A superoxide-dependent, iron-catalyzed peroxidation system was employed with xanthine oxidase plus hypoxanthine plus ferric iron-adenosine diphosphate complex as free radical generator. Purified cardiac membrane phospholipid (as liposomes) was the peroxidative target, and 15-hydroperoxy-eicosatetraenoic acid was used as a standard lipid hydroperoxide. Exposure of myocardial phospholipid to free radical generator at physiological pH (7.4) and temperature (37°C) was found to support not only phospholipid peroxidation, but also rapid lipid hydroperoxide breakdown and consequent malondialdehyde formation during peroxidation. Under lipid peroxidation conditions, oxidative injury to the phospholipid polyunsaturated fatty acids required superoxide radical and ferric iron-adenosine diphosphate complex, whereas 37°C temperature and trace iron were sufficient for lipid hydroperoxide decomposition to malondialdehyde. Harsh thiobarbituric acid-test conditions following peroxidation were not mandatory for either lipid hydroperoxide breakdown or thiobarbituric acid-reactive malondialdehyde formation. However, hydroperoxide decomposition that had begun in the peroxidation reaction could be completed during a subsequent thiobarbituric acid test in which no lipid autoxidation took place. Iron was more critical than heat in promoting the observed hydroperoxide decomposition to malondialdehyde during the lipid peroxidation reaction at 37°C and pH 7.4. These data demonstrate that the radical generator, at physiological pH and temperature, serves a dual role as both initiator of membrane phospholipid peroxidation and promotor of lipid peroxide breakdown and thiobarbituric acid-reactive malondialdehyde formation. Consequently, peroxidation reaction conditions can directly influence lipid hydroperoxide decomposition, malondialdehyde production and system thiobarbituric acid-reactivity. In vivo, decomposition of lipid peroxides to malondialdehyde during radical-mediated, metal-catalyzed membrane peroxidation may represent an integral component of oxidative tissue injury rather than a mere consequence of hydrolyzing the peroxidized biological sample in a thiobarbituric acid test.

Cardiac membrane vitamin E and malondialdehyde levels in heart muscle of normotensive and spontaneously-hypertensive rats

The vitamin E (α-tocopherol) and free and bound malondialdehyde (MDA) in ventricular heart muscle and myocardial membrane from Wistar-Kyoto (W/K) normotensive and spontaneously hypertensive (SH) rats have been measured directly by high performance liquid chromatography (HPLC). Thiobarbituric acid-reactive substance (TBA-RS) in the myocardium and heart-muscle membrane of the two strains was also quantified by a colorimetric TBA test. It was found that SH-rat myocardium and myocardial membrane contained more than 3-fold less α-tocopherol than did heart muscle and cardiac membrane of the normotensive rat. Coincident with this relative vitamin E deficiency were several-fold greater amounts of MDA and TBA-RS in SH-rat myocardium and myocardial membrane. Most (87%) of the MDA in SH-rat heart muscle, but only 40% in W/K-rat heart muscle, was free (i.e., unbound). These results offer direct evidence that SH-rat myocardium is vitamin E-deficient and highly peroxidative, relative to cardiac muscle of the normotensive W/K parent strain. The lower vitamin E content of SH-rat myocardium is particularly striking, because SH-rat myocardial membrane was found to contain ∼35% more phospholipid than myocardial membrane in the W/K rat. Although the amounts of myocardial TBA-RS are greater in the SH strain, they do not reflect the actual MDA profiles of the heart muscles or the heart membranes and cannot be used as a quantitative index of cardiac oxidative-injury status due to non-MDA TBA-RS in both strains.

Oxidative injury to myocardial membrane: Direct modulation by endogenous α-tocopherol

Abstract Peroxidation of myocardial-membrane phospholipid is considered an important pathogenic component of heart muscle damage in ischemia and reperfusion. The extent to which membrane α-tocopherol (vitamin E) in the heart can modulate such damage and protect against it is a matter of controversy. The relative α-tocopherol deficit of spontaneously-hypertensive (SH) rat myocardium as compared to the myocardium of the Wistar-Kyoto ( W K ) normotensive parent strain prompted use of these animals to identify and characterize any protective antiperoxidant role of endogenous, myocardial-membrane α-tocopherol. With exposure to a superoxide- and iron-containing initiator of peroxidation, the membrane complements from the ventricular myocardia of the SH rat and the W K parent strain were found to have very different peroxidative-injury profiles. SH-rat myocardial membrane demonstrated a marked sensitivity to peroxidation as reflected in the acute onset and rapid progression of phospholipid damage. The greater susceptibility of SH-rat myocardial membrane to free-radical attack could not be explained by inter-strain compositional differences in membrane polyunsaturated fatty acids or fatty aldehydes. Rather, the basis for the enhanced peroxidation was identified as the 3-fold lower α-tocopherol content of SH-rat myocardial membrane with respect to the heart-muscle membrane from the normotensive animal. The relative α-tocopherol deficit not only increased the susceptibility of SH-rat cardiac membrane to damage under pro-oxidant conditions, but also reduced the efficacy of exogenously supplied antioxidant intervention. These findings demonstrate that membrane α-tocopherol tone is a critical protectant of myocardial phospholipid against oxidative injury and acts as a determinant of the course of heart-membrane peroxidative damage.

Influence of cardioprotective cyclooxygenase and lipoxygenase inhibitors on peroxidative injury to myocardial-membrane phospholipid

Oxygenase-catalyzed and non-enzymatic polyunsaturated fatty acid peroxidations have potential pathogenic roles in ischemic-reperfusion damage to the myocardium. Certain oxygenase inhibitors protect heart muscle from irreversible ischemic injury, and some antiperoxidants can inhibit oxygenase enzymes. We investigated the antiperoxidative abilities of eight anti-ischemic, cardioprotective oxygenase inhibitors to prevent myocardial-membrane phospholipid peroxidation through superoxide-driven, iron-promoted reactions with xanthine oxidase as the source of superoxide. Flurbiprofen, ibuprofen, and REV-5901-5 did not affect peroxidation at concentrations up to 1000 microM. BW755C, AA-861, nafazatrom, dipyridamole, and propyl gallate did protect and cardiac lipids against oxidative injury in a concentration-dependent manner with respective and antiperoxidant IC50 values (concentrations at which peroxidation was inhibited by 50%) of 0.22, 1.25, 3.0, 3.6 and 50 microM. Catechin and phenidone, known oxygenase inhibitors not yet evaluated as anti-ischemic agents, were also found to be antiperoxidants at low micromolar concentrations. Four cyclooxygenase inhibitors ineffective against myocardial infarction (aspirin, indomethacin, naproxen, and sulfinpyrazone) evidenced no antiperoxidant properties at concentrations up to 500 microM. The oxygenase inhibitor-antiperoxidants identified could neither quench superoxide radical nor inhibit xanthine oxidase. However, they were able to interrupt the propagation of an on-going peroxidation reaction. Their antiperoxidant profiles resembled those of known antioxidants, such as alpha-tocopherol, which inhibit peroxidation by intercepting lipid free-radical intermediates. These data raise the possibility that at least some oxygenase inhibitors could exert cardioprotective effects by directly influencing the sensitivity of myocardial-membrane phospholipid to peroxidative injury. Consequently, recognition of the antiperoxidant properties of these agents may aid dissection of their physiological and pharmacological actions.

Novel 6-hydroxychroman-2-carbonitrile inhibitors of membrane peroxidative injury

Novel 6-hydroxychroman-2-carbonitrile compounds have been synthesized, and their antiperoxidant activity against superoxide-dependent, iron-promoted mycocardial phospholipid peroxidation has been evaluated quantitatively. With few exceptions, these compounds afforded significant, concentration-dependent antiperoxidant protection to myocardial-membrane phospholipid at sub- to low-micromolar concentrations. Structure-activity correlation demonstrated that R1-, R2-, and R3-methyl groups in the aromatic ring enhanced antiperoxidant activity, whereas hydrophobic groups at either R4 or R5 of the pyran ring compromised antiperoxidant efficacy. The most efficacious antiperoxidant synthesized contained a catechol moiety at R4 and was some 10-fold more potent than alpha-tocopherol. None of the 6-hydroxychroman-2-carbonitrile antiperoxidants scavenged superoxide or inhibited the enzymatic superoxide generator, xanthine oxidase, at effective antiperoxidant concentrations. The ability of these compounds to interrupt the propagatory phase of an on-going peroxidation reaction indicated that they acted as antiperoxidants by trapping chain-carrying lipid peroxyl radicals. Since a number of the 6-hydroxychroman-2-carbonitriles were most potent antiperoxidants than a variety of known chain-breaking compounds, this new class of phenolic antioxidants may represent a novel approach to the design of therapeutics against diseases in which lipid peroxidation is a causative factor or in which lipid peroxidases serve as mediators.

The dopamine receptor of the rat mammotroph in cell culture as a model for drug action

Abstract Dopamine can act directly on pituitary cells to inhibit prolactin release. This action can be blocked by dopamine receptor blocking drugs such as haloperidol, sulpiride and other neuroleptic agents. Comparison of the properties of the mammotroph dopamine receptor with the adenylate cyclase linked dopamine receptor of the limbic forebrain reveals some obvious differences. For example, dopamine receptor stimulants such as S-584 and lergotrile mesylate are inactive in stimulating the adenylate cyclase preparations but are potent in inhibiting pituitary prolactin secretion. Such inhibition of prolactin secretion can be reversed by haloperidol or sulpiride. In contrast to these observations, sulpiride does not block dopamine stimulation of cAMP formation. In addition, dopamine, apomorphine or lergotrile mesylate have no effect on a pituitary adenylate cyclase preparation and dopamine fails to elevate cAMP in the intact cells in culture. Despite the similarity between these two dopamine sensitive systems with respect to a number of agonists and antagonists, the exceptions described suggest that the pituitary system with further study may offer some greater reliability as a predictive test for clinically useful agents. These results also suggest that the receptors for dopamine, like that for norepinephrine, are of two types, only one of which is coupled to adenylate cyclase.

Specific binding of 1- O -alkyl-2-acetyl- sn -glycero-3-phosphocholine (platelet-activating factor) to the intact canine platelet

Binding of 3H-labeled 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor; PAF) to the intact, washed canine platelet has been defined and characterized as being specific and receptor-mediated. Under the conditions described, specific binding to 2 X 10(7) canine platelets reached saturation within 10 min at a [3H]PAF concentration of approximately 0.4 nM. Non-specific binding was accountable for, at most, some 30% of the total PAF bound at equilibrium. Above approximately 0.4 nM [3H]PAF, total binding and non-specific binding increased in parallel. Since no involvement of PAF ligand in dog platelet intermediary metabolism during the binding incubation could be demonstrated, non-specific PAF binding may reflect a partitioning of the molecule into a cellular compartment (perhaps the platelet membranes). Equilibrium analysis revealed that the canine platelet has one class of specific binding sites with a Kd of 0.63 +/- 0.02 nM PAF, a Bmax of 222 +/- 10 fmol/10(7) platelets, and, at most, 1.33 +/- 0.06 X 10(3) binding sites/platelet. [3H]PAF specific binding to the canine platelet is ligand-selective and stereo-selective, as demonstrated by the relative abilities of non-labeled PAF and various PAF analogs/metabolites to inhibit [3H]PAF specific binding in a concentration-dependent manner. The extents to which PAF and PAF analogs were able to displace specifically-bound [3H]PAF from the canine platelet correlated well with their physiological (i. e., pro-aggregatory) effects. These data offer the first quantitative description of canine platelet high-affinity PAF binding sites/receptors and link receptor-mediated PAF binding to canine platelet physiology.