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D R Janero

Publications and source records attributed to D R Janero.

At least 37 records · Page 2Linked to original sources
PubMed1993

Identification and molecular characterization of a high-affinity cardiomyocyte transforming growth factor-beta 2 receptor.

Rat neonatal heart muscle cells (cardiomyocytes) were found to express a high-affinity surface receptor for transforming growth factor-beta 2 (TGF-beta 2). Specific binding was rapid, saturable, ligand-selective, and reversible. Equilibrium binding analyses revealed that the cardiomyocyte had one class of specific binding sites with a Kd < or = 26 pM TGF-beta 2, a Bmax of approximately 9 fmol/10(6) cells, and approximately 5,000 binding sites/cardiomyocyte. Binding was selective for TGF-beta 2 in comparison to other TGF-beta isoforms and to unrelated growth factors. Affinity-binding experiments revealed three types of cardiomyocyte TGF-beta 2 binding proteins, the most prominent of which corresponded to the high-molecular mass proteoglycan. These data raise the possibility that the anti-ischemic cardioprotective effects of TGF-beta may reflect receptor-mediated signal transduction at the cardiomyocyte level.

Affinity Labels↗
PubMed1993

Isolation and characterization of AMP deaminase from mammalian (rabbit) myocardium.

AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) is a ubiquitous enzyme in eukaryotes, which may play a role in ATP catabolism during myocardial ischaemia. We report isolation of AMP deaminase from rabbit myocardium with a 19% recovery and a 650-fold enrichment, using a newly devised protocol involving sequential cation-exchange, gel-permeation and affinity chromatographies. The cardiac AMP deaminase preparation described was electrophoretically and chromatographically homogeneous and contained one unique N-terminal residue (leucine). The isolated enzyme was sensitive to various cations (K+, Mg2+, Ca2+). The pH optimum of purified cardiac AMP deaminase was 6.8, its pI was 6.5, and it displayed substrate-specificity toward 5'-AMP. The subunit molecular mass of rabbit heart AMP deaminase on SDS/PAGE (81 kDa) and the holoenzyme molecular mass as estimated by non-denaturing size-exclusion h.p.l.c. (330 kDa) indicated that the native enzyme was a tetramer. Cardiac AMP deaminase displayed a sigmoidal substrate-saturation curve in the presence of 100 mM KCl. Apparent Michaelis constants were a Km of 5.8 mM AMP and a Vmax. of 11.1 mumol/min per mg of protein. ATP and ADP were positive allosteric effectors of cardiac AMP deaminase: the apparent Km was decreased to 1.7 mM by 1.0 mM ATP. The enzyme was inhibited by GTP, coformycin, coformycin 5'-phosphate, palmitoyl-CoA, inorganic phosphate compounds, and the metal chelator o-phenanthroline. No inhibition either by product nucleotide (IMP) or by nicotinamide nucleotides was detected when these agents were examined at concentrations up to 2.5 mM. We conclude that this enzyme preparation offers a means by which the kinetic mechanism and regulation of mammalian cardiac AMP deaminase may be directly investigated.

AMP Deaminase↗
PubMed1993

Hydrogen peroxide-induced oxidative stress to the mammalian heart-muscle cell (cardiomyocyte): nonperoxidative purine and pyrimidine nucleotide depletion.

Hydrogen peroxide (H2O2) overload may contribute to cardiac ischemia-reperfusion injury. We report utilization of a previously described cardiomyocyte model (J. Cell. Physiol., 149:347, 1991) to assess the effect of H2O2-induced oxidative stress on heart-muscle purine and pyrimidine nucleotides and high-energy phosphates (ATP, phosphocreatine). Oxidative stress induced by bolus H2O2 elicited the loss of cardiomyocyte purine and pyrimidine nucleotides, leading to eventual de-energization upon total ATP and phosphocreatine depletion. The rate and extent of ATP and phosphocreatine loss were dependent on the degree of oxidative stress within the range of 50 microM to 1.0 mM H2O2. At the highest H2O2 concentration, 5 min was sufficient to elicit appreciable cardiomyocyte high-energy phosphate loss, the extent of which could be limited by prompt elimination of H2O2 from the culture medium. Only H2O2 dismutation completely prevented ATP loss during H2O2-induced oxidative stress, whereas various free-radical scavengers and metal chelators afforded no significant ATP preservation. Exogenously-supplied catabolic substrates and glycolytic or tricarboxylic acid-cycle intermediates did not ameliorate the observed ATP and phosphocreatine depletion, suggesting that cardiomyocyte de-energization during H2O2-induced oxidative stress reflected defects in substrate utilization/energy conservation. Compromise of cardiomyocyte nucleotide and phosphocreatine pools during H2O2-induced oxidative stress was completely dissociated from membrane peroxidative damage and maintenance of cell integrity. Cardiomyocyte de-energization in response to H2O2 overload may constitute a distinct nonperoxidative mode of injury by which cardiomyocyte energy balance could be chronically compromised in the post-ischemic heart.

Adenosine Triphosphate↗
PubMed1993

Radioiodination of transforming growth factor-beta (TGF-beta) in a modified Bolton-Hunter reaction system.

We have optimized the use of the Bolton-Hunter reagent to prepare 125I-labeled transforming growth factor-beta (TGF-beta). Conditions were developed to obtain monovalent modification of human-recombinant TGF-beta 2 (hrTGF-beta 2) at a basic pH necessary for efficient protein acylation (> or = 26% of theoretical) while obviating the problems of TGF-beta aggregation/precipitation. The purified Bolton-Hunter labeled hrTGF-beta 2 had a specific activity of 1.8-2.1 microCi/pmol, and the 125I label was fully acid-precipitable. [125I]hrTGF-beta 2 was electrophoretically indistinguishable from unlabeled starting material and displayed full immunoreactivity with polyclonal anti-TGF-beta 2 antibody. Both hrTGF-beta 2 and Bolton-Hunter-labeled [125I]hrTGF-beta 2 inhibited the growth of mink lung epithelial cells with equal efficacy. These data validate a modified conjugation-iodination method for TGF-beta and invite general use of the Bolton-Hunter reagent for iodination of other TGF-beta isoforms and peptides similarly susceptible to precipitation/aggregation under standard Bolton-Hunter incubation conditions.

Animals↗
PubMed1993

Hydroperoxide-induced oxidative stress alters pyridine nucleotide metabolism in neonatal heart muscle cells.

An oxidant burden established by hydrogen peroxide (H2O2) overload may elicit postischemic myocardial damage. We assess herein the influence of H2O2-induced oxidative stress on heart muscle pyridine nucleotide metabolism. Exposure of neonatal rat cardiomyocytes to 50 microM-1.0 mM H2O2 bolus rapidly shifted their pyridine-nucleotide redox balance toward oxidation. At least 30% of the observed NADPH oxidation was independent of glutathione cycle activity and appeared chemical in nature with H2O2 itself, and not a radical metabolite, acting as oxidant. Cell exposure to H2O2 also depleted cardiomyocyte pyridine nucleotides as a consequence of enhanced utilization. The oxidative stress activated one major route of pyridine nucleotide catabolism (i.e., protein ADP-ribosylation) without acute inhibitory effect upon the other (cleavage by NAD glycohydrolase). The limited NAD sparing by metal chelators and inhibitors of ADP-ribosylation reflected pyridine nucleotide utilization for repair of single-strand DNA breaks caused by hydroxyl-like radicals formed intracellularly through iron-dependent H2O2 reduction. Cardiomyocyte NAD depletion during H2O2-induced oxidative stress was independent of cell integrity and lipid peroxidation. The NAD lost after a discrete H2O2 "pulse" was only partly replenished over a 24-h postinjury period. These data demonstrate that cardiomyocyte pyridine nucleotide metabolism is a nonperoxidative injury target that is chronically affected by H2O2 overload. Derangement of myocardial pyridine nucleotide pools due to oxidative stress may contribute to ischemic heart injury in vivo by interfering with cardiac hydrogen metabolism and redox balance.

Animals↗
PubMed1993

Adenosine deaminase inhibitors attenuate ischemic injury and preserve energy balance in isolated guinea pig heart.

We investigated the effect of the adenosine deaminase inhibitors erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and coformycin on high-energy phosphate metabolism, tissue nucleotides and nucleosides, and recovery of contractile function in isolated, perfused guinea pig hearts. EHNA and coformycin (10 microM) improved postischemic recovery of contractile function approximately 85% and enhanced coronary flow rate in reperfused tissue approximately 40%. The protective effect of EHNA on recovery of contractile function was concentration dependent. Although adenosine (10 microM) increased coronary flow rate on reperfusion approximately twofold over vehicle, it failed to improve postischemic recovery of contractile function. EHNA and coformycin preserved cardiac ATP levels and increased endogenous tissue adenosine during ischemia. During reperfusion, these agents enhanced recovery of high-energy phosphates approximately twofold and potentiated adenosine release into the perfusate with concentration dependency. Furthermore, EHNA and coformycin reduced the extent of myocardial ischemia-reperfusion injury, as indicated by the approximately 55% reduction in creatine phosphokinase release. We conclude that inhibitors of adenosine deaminase attenuate myocardial ischemic injury and improve postischemic recovery of contractile function and metabolism through endogenous myocardial adenosine enhancement and ATP preservation.

Adenine↗
PubMed1992

Application of solid-phase extraction on anion-exchange cartridges to quantify 5'-nucleotidase activity.

The enzyme 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) catalyzes a critical reaction in intermediary metabolism, the phosphohydrolysis of nucleoside 5'-monophosphates to their corresponding nucleosides. We have evaluated solid-phase extraction on pre-packed anion-exchange cartridges as a chromatographic technique with which 5'-nucleotidase activity may be detected and quantified. Chromatographic conditions were established whereby substrate nucleotide was rapidly and completely separated from its corresponding nucleoside by solid-phase extraction. Both analytes were recovered quantitatively, without loss or degradation. This chromatographic system was integrated into a discontinuous radiochemical assay for 5'-nucleotidase which enabled both substrate utilization and product formation to be assessed simultaneously. Enzyme reaction samples could be analyzed directly for 5'-nucleotidase activity without any pre-chromatography preparation. The high capacity of the solid-phase cartridges and the inability of 5'-nucleotidase to enter the packing bed during analyte elution facilitated termination of the enzyme reaction by applying the entire reaction mixture to the cartridge. Loaded cartridges could then be stored at 4 degrees C prior to chromatography and subsequently batch-eluted. The excellent resolution between substrate and product in solid-phase extraction and the sensitivity of radioisotopic counting enabled detection/quantification of low tissue levels of 5'-nucleotidase in conjunction with ancillary assays for secondary enzyme reactions with the potential to elicit the artifactual loss of 5'-nucleotidase substrate/product when crude biological preparations are examined for 5'-nucleotidase activity. Our results demonstrate that solid-phase extraction on anion-exchange cartridges with elution solvents of appropriate pH offers several unique advantages for 5'-nucleotidase determination.

5'-Nucleotidase↗
PubMed1991

Hydrogen peroxide-induced oxidative stress to the mammalian heart-muscle cell (cardiomyocyte): lethal peroxidative membrane injury.

Oxidative stress induced by hydrogen peroxide (H2O2) may contribute to the pathogenesis of ischemic-reperfusion injury in the heart. For the purpose of investigating directly the injury potential of H2O2 on heart muscle, a cellular model of H2O2-induced myocardial oxidative stress was developed. This model employed primary monolayer cultures of intact, beating neonatal-rat cardiomyocytes and discrete concentrations of reagent H2O2 in defined, supplement-free culture medium. Cardiomyocytes challenged with H2O2 readily metabolized it such that the culture content of H2O2 diminished over time, but was not depleted. The consequent H2O2-induced oxidative stress caused lethal sarcolemmal disruption (as measured by lactate dehydrogenase release), and cardiomyocyte integrity could be preserved by catalase. During oxidative stress, a spectrum of cellular derangements developed, including membrane phospholipid peroxidation, thiol oxidation, consumption of the major chain-breaking membrane antiperoxidant (alpha-tocopherol), and ATP loss. No net change in the protein or phospholipid contents of cardiomyocyte membranes accompanied H2O2-induced oxidative stress, but an increased turnover of these membrane constituents occurred in response to H2O2. Development of lethal cardiomyocyte injury during H2O2-induced oxidative stress did not require the presence of H2O2 itself; a brief "pulse" exposure of the cardiomyocytes to H2O2 was sufficient to incite the pathogenic mechanism leading to cell disruption. Cardiomyocyte disruption was dependent upon an intracellular source of redox-active iron and the iron-dependent transformation of internalized H2O2 into products (e.g., the hydroxyl radical) capable of initiating lipid peroxidation, since iron chelators and hydroxyl-radical scavengers were cytoprotective. The accelerated turnover of cardiomyocyte-membrane protein and phospholipid was inhibited by antiperoxidants, suggesting that the turnover reflected molecular repair of oxidized membrane constitutents. Likewise, the consumption of alpha-tocopherol and the oxidation of cellular thiols appeared to be epiphenomena of peroxidation. Antiperoxidant interventions coordinately abolished both H2O2-induced lipid peroxidation and sarcolemmal disruption, demonstrating that an intimate pathogenic relationship exists between sarcolemmal peroxidation and lethal compromise of cardiomyocyte integrity in response to H2O2-induced oxidative stress. Although sarcolemmal peroxidation was causally related to cardiomyocyte disruption during H2O2-induced oxidative stress, a nonperoxidative route of H2O2 cytotoxicity was also identified, which was expressed in the complete absence of cardiomyocyte-membrane peroxidation. The latter mode of H2O2-induced cardiomyocyte injury involved ATP loss such that membrane peroxidation and cardiomyocyte disruption on the one hand and cellular de-energization on the other could be completely dissociated.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗
PubMed1991

Therapeutic potential of vitamin E against myocardial ischemic-reperfusion injury.

Myocardial ischemia is a disease process characterized by reduced coronary flow such that the supply of nutritive blood to heart muscle (myocardium) is insufficient for normal myocardial aerobic metabolism. Prompt reestablishment of coronary flow by invasive and noninvasive clinical procedures is the most direct and effective means of limiting myocardial damage in ischemic heart disease patients, although reperfusion carries with it an injury component which may reflect, at least to some degree, the toxic effects of partially reduced oxygen species and their participation in degenerative cellular processes such as membrane lipid peroxidation. Vitamin E, a lipophilic, chain-breaking antioxidant, is a prominent membrane constituent in heart muscle, where it modulates/regulates various aspects of heart muscle-cell metabolism and function. Vitamin E's beneficial effects against experimentally induced oxidative damage to the heart, along with inverse epidemiological correlations between plasma vitamin E level and either anginal pain or mortality due to ischemic heart disease, suggest that vitamin E might have protective and therapeutic roles against myocardial ischemic-reperfusion injury. Laboratory investigations aimed at addressing this possibility have demonstrated that vitamin E supplementation protects isolated hearts against ischemic-reperfusion injury, and relatively more inconsistent and limited data document cardioprotective effects of vitamin E in some animal models of myocardial ischemia-reperfusion, especially when administered prior to the ischemic period. Clinical attempts to establish whether vitamin E has therapeutic benefit in ischemic heart disease patients remain inconclusive, having relied upon a variety of nonuniformly controlled protocols and a single, rather subjective endpoint (anginal pain). Consequently, although laboratory data constitute a conceptual context for and indirect support of the idea that vitamin E could be a cardioprotectant against ischemic-reperfusion injury, compelling clinical evidence regarding vitamin E's therapeutic potential in the ischemic heart-disease patient is lacking. Elective coronary revascularization would appear to provide an attractive clinical setting for evaluating the therapeutic efficacy of vitamin E in the context of cardiac ischemia-reperfusion. Further biochemical work would still be required to define how vitamin E exerts any cardioprotective effect observed in these patients.

Animals↗
PubMed1991

Therapeutic potential of vitamin E in the pathogenesis of spontaneous atherosclerosis.

Spontaneous atherosclerosis is largely an occlusive disease of medium-size arteries whose progression in a hyperlipidemic environment reflects chronic interactions among injury stimuli to the vessel wall and "responses to injury" by vascular tissue and certain blood components. Development of vessel lesions in animal models of spontaneous atherosclerosis and (at least in principle) in man largely reflects responses of three major cell types (vascular endothelial cells, vascular smooth muscle cells, monocytes-macrophages) as well as the content and distribution of lipids among various lipoprotein subclasses and the increased atherogenicity of modified (e.g., oxidized) lipoproteins. The severe clinical complications associated with spontaneous atherosclerosis, along with its rather common incidence in man, have focused attention on the prevention and therapy of this vascular disease state. Some pharmacological studies in animal models of spontaneous atherosclerosis and some retrospective epidemiological studies in man suggest that vitamin E, the principal (if not sole) lipid-soluble chain-breaking tissue antioxidant, might have therapeutic benefit as an antiatherosclerotic agent. This suggestion gains support from a variety of compelling in vitro evidence demonstrating direct influences of vitamin E on cells and lipoproteins likely involved in the pathogenesis of spontaneous atherosclerosis. Biochemical and cellular data indicate that the potential antiatherogenic activity of vitamin E could reflect its activities as a regulator of endothelial, smooth muscle, or monocyte-macrophage function, an inhibitor of endothelial membrane lipid peroxidation, a modulator of plasma lipid levels and lipid distribution among circulating lipoproteins, and a preventor of lipoprotein oxidative modification. On the other hand, there is a comparative lack of conclusive evidence from animal models regarding: (a) the importance to atherogenesis of vascular and cellular processes modulated by vitamin E; (b) the influence of vitamin E on these processes in vivo and, consequently, on the initiation/progression of spontaneous atherosclerosis. Therefore, pharmacologic investigation of vitamin E (and synthetic, vitamin E-like antioxidants) in nutritional and hyperlipidemic animal models of spontaneous atherosclerosis is required to establish whether any atherosclerotic impact is associated with vitamin E and, if so, what the mechanistic basis of the therapeutic benefit is. Such a line of experimental inquiry should also increase our understanding of the pathogenesis of atherosclerotic vessel disease per se.

Animals↗
PubMed1991

Examination of two small-molecule antiperoxidative agents in a rabbit model of postischemic myocardial infarction.

Peroxidation of membrane phospholipid may be a causal contributor to the development of irreversible postischemic myocardial injury. In this study, two small-molecule antiperoxidative agents were tested for their ability to salvage reperfused rabbit myocardium and reduce infarct size as assessed by direct histological evaluation of hearts following a 30-min occlusion of a coronary arterial branch and a 72-h reperfusion period. The compounds tested are novel analogs of alpha-tocopherol (vitamin E), and share with the parent compound an ability to scavenge the peroxyl radicals that propagate and amplify lipid peroxidation initiated by partially reduced oxygen; however, the new analogs are significantly more potent peroxyl-radical scavengers that alpha-tocopherol. Each antiperoxidant (3 mg/kg) was administered by intravenous bolus injection in two stages: 20% of the total dose was given 15 min before occlusion, and the remaining 80% was given 5 min before reperfusion. The results revealed that neither antiperoxidant offered a sustained reduction in infarct size (expressed as a percentage of the region at risk) as compared to a nontreated vehicle control. The implications of the present study with respect to the purported cardioprotective effects of antiperoxidants in the setting of ischemia reperfusion and the pathogenic role of lipid peroxidation in the postischemic heart are discussed.

Animals↗
PubMed1991

Pharmacology of a potent platelet-activating factor antagonist: Ro 24-4736.

Ro 24-4736, (5-(3-[4-(2-chlorophenyl)-9-methyl-6H-thieno[3,2-f] [1,2,4]triazolo[4,3-a][1,4]diazepin-2-yl]-2-propynyl)phenanthri din- 6(5H)-one), has been identified as a potent, selective, p.o.-active platelet-activating factor (PAF) antagonist with a long duration of action. In vitro, Ro 24-4736 competes with [3H]PAF for its receptor site on dog platelets with an IC50 of 9.8 +/- 1.0 nM and selectively inhibits PAF-induced aggregation of guinea pig, dog and human platelets with concentration dependence. Ro 24-4736 dose-dependently inhibits in vivo bronchoconstriction (ID50 of 0.006-mg/kg p.o.) and ex vivo platelet aggregation (ID50 of 0.004 mg/kg p.o.) induced by PAF in guinea pigs. Time course studies show complete blockade of PAF-induced platelet aggregation (ex vivo) up to 8 hr after a single p.o. dose of 0.03 mg/kg as well as a long duration of action in vivo (30 hr). The in vivo PAF antagonistic activity is specific because, even at high p.o. doses (up to 10 mg/kg), Ro 24-4736 shows no inhibitory activity toward the bronchoconstrictor effects of leukotriene D4 or histamine. In comparison with other PAF antagonists evaluated in this guinea pig model, Ro 24-4736 is markedly superior in terms of p.o. potency, bioavailability and p.o. duration of action. Studies were also performed with Ro 24-4736 in additional in vivo models. When administered p.o. to sensitized guinea pigs, the drug attenuates inhaled antigen-induced airway hyper-reactivity without effect on bronchoalveolar lavage leukocyte accumulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Solid-phase extraction on silica cartridges as an aid to platelet-activating factor enrichment and analysis.

Solid-phase extraction methods using pre-packed silica cartridges and various elution solvents have been developed and evaluated as chromatographic means to enrich biological lipid extracts for platelet-activating factor (PAF). The optimized procedure advanced selectively removed the major tissue/blood neutral lipids and non-choline-containing phospholipids from complex lipid mixtures and yielded thereby a choline phospholipid fraction markedly enriched in bioactive PAF. Some tested solid-phase extraction procedures, while capable of resolving choline phospholipids from other polar and non-polar species, were detrimental to PAF's bioactivity and evidenced considerable loss or degradation of this analyte. It is concluded that, with solvents of appropriate composition, strength and polarity, solid-phase extraction on silica cartridges has several unique advantages over conventional thin-layer and column chromatographic methods presently in use for PAF enrichment from biological sources.

Animals↗
PubMed1990

Malondialdehyde and thiobarbituric acid-reactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury.

Increasing appreciation of the causative role of oxidative injury in many disease states places great importance on the reliable assessment of lipid peroxidation. Malondialdehyde (MDA) is one of several low-molecular-weight end products formed via the decomposition of certain primary and secondary lipid peroxidation products. At low pH and elevated temperature, MDA readily participates in nucleophilic addition reaction with 2-thiobarbituric acid (TBA), generating a red, fluorescent 1:2 MDA:TBA adduct. These facts, along with the availability of facile and sensitive methods to quantify MDA (as the free aldehyde or its TBA derivative), have led to the routine use of MDA determination and, particularly, the "TBA test" to detect and quantify lipid peroxidation in a wide array of sample types. However, MDA itself participates in reactions with molecules other than TBA and is a catabolic substrate. Only certain lipid peroxidation products generate MDA (invariably with low yields), and MDA is neither the sole end product of fatty peroxide formation and decomposition nor a substance generated exclusively through lipid peroxidation. Many factors (e.g., stimulus for and conditions of peroxidation) modulate MDA formation from lipid. Additional factors (e.g., TBA-test reagents and constituents) have profound effects on test response to fatty peroxide-derived MDA. The TBA test is intrinsically nonspecific for MDA; nonlipid-related materials as well as fatty peroxide-derived decomposition products other than MDA are TBA positive. These and other considerations from the extensive literature on MDA. TBA reactivity, and oxidative lipid degradation support the conclusion that MDA determination and the TBA test can offer, at best, a narrow and somewhat empirical window on the complex process of lipid peroxidation. The MDA content and/or TBA reactivity of a system provides no information on the precise structures of the "MDA precursor(s)," their molecular origins, or the amount of each formed. Consequently, neither MDA determination nor TBA-test response can generally be regarded as a diagnostic index of the occurrence/extent of lipid peroxidation, fatty hydroperoxide formation, or oxidative injury to tissue lipid without independent chemical evidence of the analyte being measured and its source. In some cases, MDA/TBA reactivity is an indicator of lipid peroxidation; in other situations, no qualitative or quantitative relationship exists among sample MDA content, TBA reactivity, and fatty peroxide tone. Utilization of MDA analysis and/or the TBA test and interpretation of sample MDA content and TBA test response in studies of lipid peroxidation require caution, discretion, and (especially in biological systems) correlative data from other indices of fatty peroxide formation and decomposition.

Animals↗
PubMed1990

Production and release of platelet-activating factor by the injured heart-muscle cell (cardiomyocyte).

The potential of the injured heart-muscle cell (cardiomyocyte) to produce platelet-activating factor (PAF) has been evaluated. Beating rat myocytes in primary monolayer culture were used as the study-object, and metabolic inhibition served as the injury stimulus. Coincident with the development of irreversible injury, myocytes evidenced a rapid, net production of PAF which was sustained for some 3-6 h and reached, maximally, low-ng levels within approximately 0.5 h of injury. In the presence of serum, much of the PAF was released from the injured myocytes, whereas under serum-free conditions the PAF remained largely intracellular. Cardiomyocyte-derived PAF was pro-aggregatory to platelets and displayed the chromatographic properties and chemical reactivity of authentic PAF. However, PAF itself did not elicit myocyte injury, and a specific PAF antagonist could not attenuate the myocyte damage associated with metabolic inhibition. These results suggest that the cardiomyocyte may constitute a cellular source of PAF during heart injury, although the cell itself is not directly responsive to PAF.

Adenosine Triphosphate↗
PubMed1989

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.

Aspirin↗
PubMed1989

Nonesterified fatty acid accumulation and release during heart muscle-cell (myocyte) injury: modulation by extracellular "acceptor".

Long-chain nonesterified fatty acid (NEFA) accumulation in the heart muscle cell (myocyte) and NEFA release to the extracellular milieu are considered contributors to the pathogenesis of myocardial injury in a number of cardiovascular disease states. Reported here is a study of the factors which influence and control the interactions among NEFA formation, intracellular NEFA accumulation, and NEFA release to the extracellular compartment by the irreversibly injured myocyte. Under conditions of metabolic inhibition, neonatal rat myocytes in primary monolayer culture became virtually depleted of ATP within 8 h. The metabolically inhibited myocytes evidenced membrane phospholipid degradation and a resultant net accumulation of NEFA produced thereby in the extracellular medium. However, under conditions of nutrient deprivation, the injured myocytes retained the NEFA produced from phospholipid catabolism intracellularly and did not release it to the culture medium, although the extent of myocyte ATP depletion was the same as it had been from metabolic inhibition. Serum could elicit, in a concentration-dependent fashion, the quantitative release of NEFA from metabolically inhibited myocytes to the culture medium but did not influence the net production of NEFA by the injured cells. Similarly, NEFA release from nutrient-deprived myocytes incubated in serum-free, substrate-free medium or in physiological buffer could be induced by supplementing the medium or buffer with bovine serum albumin (BSA), and the extent of NEFA release, but not NEFA formation, was dependent upon the extracellular BSA concentration. No manipulations to media other than changing their serum content or supplementing them with BSA were found to influence the disposition of NEFA produced during phospholipid catabolism in the irreversibly injured, ATP-depleted myocyte. Therefore, although progressive metabolic compromise in the myocyte was correlated with increasing, net NEFA formation, the distribution of the NEFA between the intracellular and the extracellular compartments was not determined by the magnitude of ATP loss or by the nature or duration of at least two injury stimuli, metabolic inhibition and nutrient deprivation. Rather, the net release of NEFA from the ATP-depleted myocyte to the culture medium and the consequent reduction of intracellular myocyte NEFA overload were critically and causally dependent upon the presence and concentration of extracellular NEFA "acceptor". The influence of acceptor on the mobilization of NEFA from the injured myocyte has implications regarding the use of NEFA release as an index of myocyte pathology and could serve to modify the progression and extent of myocardial injury in vivo.

Adenosine Triphosphate↗