Bone regeneration is influenced by mesenchymal stromal cells (MSCs) and mechanical conditions. How healing outcome and mechanical stability are linked on the cellular level, however, remains elusive. Cyclic-compressive loading of MSCs affects the expression of molecules involved in angiogenesis and matrix assembly, but also reduces the expression of CD73, an ecto-5'-nucleotidase, which plays a crucial role in extracellular adenosine generation. Although, for almost 20 years, CD73 has been a major cell surface marker defining MSCs, little is known about its function in these cells. Therefore, the aim of this study was to determine the putative involvement of CD73 in MSC differentiation after cyclic-compressive loading. After cultivation in appropriate differentiation media, chondrogenic differentiation ability was significantly increased in loaded MSCs, hence following current models. Through treatment with the CD73 inhibitor adenosine 5'-(α, β-methylene) diphosphate, chondrogenic matrix deposition was further increased; in contrast, mineral matrix deposition and expression of osteogenic markers was reduced. One major signal transduction pathway, which is activated via CD73-mediated adenosine, is the adenosine receptor pathway. Thus, the adenosine receptor expression pattern was investigated. MSCs expressed the four known adenosine receptors at the mRNA level. After mechanical stimulation of MSCs, Adora2a was down-regulated. These data point towards a role of CD73 in MSC differentiation possibly via A2AR signalling, which is mutually regulated with CD73. In conclusion, the findings of this study suggest that CD73 is another regulatory factor in osteo-/chondrogenic differentiation of MSCs and may provide a - thus far underestimated - therapeutic target to guide bone regeneration.

BACKGROUND

Salvianolic acid A (SAA), the water-soluble phenolic acids in Salvia miltiorrhiza, has shown the most potent bioactivities, including protection against cerebral lesion, defense from oxidative damage and improvement of remembrance. In the present study, we studied the antiplatelet and antithrombotic effects of a newly synthesized SAA with different methods both in vitro and in vivo.

METHODS

We tested the effect of antithrombotic activity of SAA in arterio-venous shunt model. The effects of SAA on adenosine diphosphate (ADP)-, Thrombin-, Arachidonic acid- induced rat platelets aggregation were tested both in vivo and in vitro. The activity of SAA on washed human platelet aggregation was determined by ADP stimulation. We also evaluated its property of modulation of hemorheology, assessed its bleeding side effect by measuring coagulation parameters after intravenous administration for 5 days and investigated the potential mechanisms underlying such activities.

CONCLUSIONS

In vivo, SAA significantly reduced thrombus weight in the model of arterio-venous shunt. Meanwhile, SAA increased plasma cAMP level determined by radioimmunoassay in the same model. Intravenously administrated SAA (2.5-10 mg/kg) inhibited platelet aggregation induced by ADP in a dose-dependent manner. Notably, SAA did not affect coagulation parameters in rats after intravenous administration SAA for successive 5 days. In vitro, pretreatment with SAA on washed rat and human platelets significantly inhibited various agonists stimulated platelet aggregation and caused an increase in cAMP level in platelets activated by ADP. These findings support our hypothesis that SAA possesses antithrombotic activities. The antithrombotic effect might be related to its antiplatelet action and ability to modulate hemorheology without affecting coagulation system. The mechanisms underlying such activities may involve the induction of cAMP.

Sickle erythrocyte (RBC) membranes were previously shown to manifest increased Fenton activity (iron-dependent, peroxide-driven formation of hydroxyl radical [.OH]) compared with normal RBC membranes, but the nature of the catalytic iron was not defined. We now find that sickle membranes exposed to superoxide (.O2-) and hydrogen peroxide (H2O2) have three distinct iron compartments able to act as Fenton catalysts: preexisting free iron, free iron released during oxidant stress, and a component that cannot be chelated with deferoxamine (DF). In a model system, addition of iron compounds to normal ghosts showed that free heme, hemoglobin, Fe/adenosine diphosphate (ADP), and ferritin all catalyze .OH production; concurrent inhibition studies using DF documented that the unchelatable Fenton component is free heme or hemoglobin. During exposure to peroxide only, the iron in sickle membranes was unable to act as a Fenton catalyst without addition of a reducing agent. At physiologic concentrations, both ascorbate and glutathione restored Fenton activity. Lipid peroxidation studies showed that at physiologic levels ascorbate acts primarily as an antioxidant; however, as pharmacologic levels are reached, its pro-oxidant effects predominate. This study elucidates the catalytic ability of the iron compartments in the sickle cell membrane, the importance of which relates to the potential role of .OH in membrane damage. It also illustrates the potential participation of cytoplasmic reducing agents in this process, which may be especially relevant in the context of administration of supraphysiologic doses of ascorbate to sickle cell patients.

We have reported previously two siblings with gout and uric acid lithiasis associated with excessive purine production. In the erythrocytes of these patients, phosphoribosylpyrophosphate (PRPP) synthetase exhibited resistance to feedback-inhibition by normal cell constituents such as guanosine-5'-diphosphate (GDP) and adenosine-5'-diphosphate (ADP), resulting in superactivity of the mutant enzyme and consequently in increased PRPP content and availability for nucleotide synthesis. Erythrocyte PRPP content and availability were normal in the propositus' parents, his healthy brother and three sons, and they all had normal serum level and urinary excretion of uric acid, except for the mother who was hyperuricosuric. To further characterize this mutation we studied PRPP and purine metabolism in cultured fibroblasts of the affected family. PRPP synthetase in dialyzed lysates of fibroblasts from the propositus and his mother exhibited increased specific activity, more markedly at low inorganic phosphate concentration, and decreased sensitivity to inhibition by ADP and GDP, PRPP content and availability and the rate of de novo purine nucleotide synthesis were markedly increased in the fibroblasts of the propositus and to a lesser extent in the fibroblasts of his mother but were normal in the fibroblasts of the other family members investigated. The fibroblast studies demonstrate the following sequence of abnormalities: feedback-resistance of PRPP synthetase; superactivity of this enzyme in normal physiological milieu; increased availability of PRPP; and increased de novo synthesis of purine nucleotides. The pattern of inheritance of this disorder is compatible with both an X-linked recessive and autosomal dominant traits.

The major clinical indication for antiplatelet therapy has been the prevention of arterial thrombosis. Arterial thrombi are composed of predominantly platelets formed under conditions of elevated shear stress at sites of atherosclerotic vascular injury and disturbed blood flow. Aspirin, the prototype antiplatelet agent, has been in clinical use as an antithrombotic for almost a half century. However, clinical trials have exposed the limitations of aspirin, and there has been considerable recent progress in the development of more effective antiplatelet agents. These newer agents are rationally based on interrupting specific sites in the sequence of platelet activation. Inhibitors of the initial step of platelet adhesion remain experimental. Inhibitors of specific platelet agonist-receptor interactions include antithrombins, thromboxane A2 receptor antagonists, and adenosine diphosphate (ADP) receptor blockers including ticlopidine and clopidogrel. Inhibitors of arachidonic acid metabolism and thromboxane A2 include omega-3 fatty acids, aspirin and other nonsteroidal antiinflammatory drugs that inhibit cyclooxygenase, and thromboxane synthase inhibitors. The clinical efficacy of many of these agents may be limited by their actions, which are restricted to single, specific platelet receptors or metabolic pathways. Global interruption of the final step of platelet aggregation can be achieved with monoclonal antibodies and RGD (arginine-glycine-aspartic acid) analogs that block ligand binding to the platelet glycoprotein IIb/IIIa complex. Initial clinical trials with these novel agents have demonstrated superior efficacy in preventing reocclusion and restenosis following coronary angioplasty and atherectomy.

Both isolated brain mitochondria and mitochondria in intact neurons are capable of accumulating large amounts of calcium, which leads to formation in the matrix of calcium- and phosphorus-rich precipitates, the chemical composition of which is largely unknown. Here, we have used inhibitors of the mitochondrial permeability transition (MPT) to determine how the amount and rate of mitochondrial calcium uptake relate to mitochondrial morphology, precipitate composition, and precipitate retention. Using isolated rat brain (RBM) or liver mitochondria (RLM) Ca(2+)-loaded by continuous cation infusion, precipitate composition was measured in situ in parallel with Ca(2+) uptake and mitochondrial swelling. In RBM, the endogenous MPT inhibitors adenosine 5'-diphosphate (ADP) and adenosine 5'-triphosphate (ATP) increased mitochondrial Ca(2+) loading capacity and facilitated formation of precipitates. In the presence of ADP, the Ca/P ratio approached 1.5, while ATP or reduced infusion rates decreased this ratio towards 1.0, indicating that precipitate chemical form varies with the conditions of loading. In both RBM and RLM, the presence of cyclosporine A in addition to ADP increased the Ca(2+) capacity and precipitate Ca/P ratio. Following MPT and/or depolarization, the release of accumulated Ca(2+) is rapid but incomplete; significant residual calcium in the form of precipitates is retained in damaged mitochondria for prolonged periods.

OBJECTIVE

The purpose of this study was to investigate whether omega-3 polyunsaturated fatty acids (PUFAs) are able to modify platelet responsiveness to dual antiplatelet therapy in stable coronary artery disease patients undergoing percutaneous coronary intervention (PCI).

BACKGROUND

Although previous studies have suggested antiplatelet properties of omega-3 polyunsaturated fatty acids, it is unknown whether they can enhance platelet inhibition on standard aspirin and clopidogrel treatment.

METHODS

The OMEGA-PCI (OMEGA-3 Fatty Acids After PCI to Modify Responsiveness to Dual Antiplatelet Therapy) study was an investigator-initiated, prospective, single-center, double-blind, placebo-controlled, randomized study. Patients receiving standard dual antiplatelet therapy (aspirin 75 mg/day and clopidogrel 600 mg loading dose followed by 75 mg/day) were randomly assigned to receive the addition of 1 g of omega-3 ethyl esters (n = 33) or placebo (n = 30) for 1 month. Platelet function was measured serially by light transmission aggregometry (adenosine diphosphate and arachidonic acid [AA] were used as agonists) and assessment of the phosphorylation status of the vasodilator-stimulated phosphoprotein at baseline, 12 h, 3 to 5 days, and 30 days after randomization.

RESULTS

The P2Y(12) reactivity index was significantly lower, by 22.2%, after 1 month of treatment with omega-3 polyunsaturated fatty acids compared with placebo when used in addition to dual antiplatelet therapy (p = 0.020). Maximal platelet aggregation induced by 5 and 20 micromol/l adenosine diphosphate was lower by 13.3% (p = 0.026) and 9.8% (p = 0.029), respectively, after 1 month of treatment with omega-3 polyunsaturated fatty acids compared with placebo. Platelet aggregation after AA stimulation was low and did not change significantly throughout the study. There were no cases of aspirin resistance during follow-up that was suggestive of good compliance with the medication.

CONCLUSIONS

The addition of omega-3 ethyl esters to the combination of aspirin and clopidogrel significantly potentiates platelet response to clopidogrel after percutaneous coronary intervention.

Rottem, Shlomo (Hebrew University, Jerusalem, Israel), and Shmuel Razin. Adenosine triphosphatase activity of mycoplasma membranes. J. Bacteriol. 92:714-722. 1966.-Adenosine triphosphatase activity of Mycoplasma laidlawii, M. gallisepticum, and Mycoplasma sp. strain 14 was confined to the cell membrane. The enzymatic activity was dependent on magnesium, but was not activated by sodium and potassium. Ouabain did not inhibit the adenosine triphosphatase activity of the mycoplasmas, and did not interfere with the active accumulation of potassium by M. laidlawii cells. Sulfhydryl-blocking reagents and fluoride inhibited the enzymatic activity, whereas 2,4-dinitrophenol was without any effect. Membranes of M. laidlawii hydrolyzed other nucleotide triphosphates and adenosine diphosphate (ADP), but at a lower rate than adenosine triphosphate (ATP). Nucleoside-2'-(3')-phosphates, ribose-5-phosphate, glucose-6-phosphate, and pyrophosphate were not hydrolyzed by the membrane preparations. It seems that the enzyme(s) involved in ATP hydrolysis by M. laidlawii membranes is strongly bound to the membrane subunits, which would account for the failure to purify the enzyme by protein fractionation techniques. The adenosine triphosphatase activity of mycoplasma membranes resembles in its properties that of similar enzymes studied in bacteria. The mycoplasma enzyme(s) seems to differ from the adenosine triphosphatase associated with ion transport in mammalian cell membranes and from mitochondrial adenosine triphosphatase.

Excessive reactivity of blood platelets may contribute to atherosclerotic vascular disease. Hence drugs which alter platelet function may be protective. Prompted by findings that propranolol therapy normalized hyperactive platelet aggregation in patients with coronary artery disease, we studied propranolol in vitro to assess its action on platelets. At concentrations similar to those achieved in vivo (0.1-1 muM), propranolol raised the thresholds for aggregation of some normal paltelets by adenosine diphosphate (ADP). At higher concentrations (10-50 muM), propranolol abolished the second wave of platelet aggregation induced by ADP and epinephrine, and inhibited aggregation induced by collagen, thrombin, and the ionophore A23187. Propanolol blocked the release of 14C-serotonin from platelets, inhibited platelet adhesion to collagen, and interfered with clot retraction. Propranolol blocked ionophore-induced uptake of 45Ca by platelets. Inhibition appeared unrelated to beta-adrenergic blockage, as d(+) propranolol (which lacks beta-blocking activity) was equipotent with 1(-) propranolol. Moreover, practolol, a beta-blockading drug which is nonlipophilic, did not inhibit platelet function. These studies suggested that propranolol, like local anesthetics, decreased platelet responsiveness by a direct action on the platelet membrane, possibly by interfering with calcium availability. Modulation of platelet function by propranolol may occur at concentrations achieved at usual clinical doses of the drug.

Thrombin and adenosine diphosphate (ADP) supported the binding of 125I-fibrinogen to washed human platelets with similar kinetics and affinity. Platelet secretion, as measured by 14C-serotonin release, and fibrinogen binding exhibited an identical dependence on thrombin concentration. Enzymatic removal of ADP with apyrase or creatine phosphate/creatine phosphokinase (CP/CPK) from thrombin-stimulated platelets markedly inhibited 125I-fibrinogen binding, but pretreatment of platelets with CP/CPK prior to thrombin stimulation was without effect. Thus, ADP, released from the platelet, participates in the binding of fibrinogen to thrombin-stimulated platelets.

Ca2+, Mg2+-ionophores X537A and A23,187 (10(-7)-10(-6) M) induced the release of adenine nucleotides adenosine diphosphate (ADP, adenosine triphosphate (ATP), serotonin, beta-glucuronidase, Ca2+, and Mg2+ from washed human platelets. Enzymes present in the cytoplasm or mitochondria, and Zn2+ were not released. The rate of ATP and Ca2+ release measured by firefly lantern extract and murexide dye, respectively, was equivalent to that produced by the physiological stimulant thrombin. Ionophore-induced release of ADP, and serotonin was substantially (approximately 60%) but not completely inhibited by EGTA, EDTA, and high extracellular Mg2+, without significant reduction of Ca2+ release. The ionophore-induced release reaction is therefore partly dependent upon uptake of extracellular Ca2+ (demonstrated using 45Ca), but also occurs to a significant extent due to release into the cytoplasm of intracellular Ca2+. The ionophore-induced release reaction and aggregation of platelets could be blocked by prostaglandin E1 (PGE1) or dibutyryl cyclic AMP. The effects of PGE1, and N6, O2-dibutyryl adenosine 3':5'-cyclic monophosphoric acid (dibutyryl cAMP) were synergistically potentiated by the phosphodiesterase inhibitor theophylline. It is proposed that Ca2+ is the physiological trigger for platelet secretion and aggregation and that its intracellular effects are strongly modulated by adenosine 3':5'-cyclic monophosphoric acid (cyclic AMP).

Adenosine 5'-diphosphate (ADP), inorganic pyrophosphate (PPi), and adenyl-5'-yl imidodiphosphate (AMPPNP) act as competitive inhibitors of the ATPase of myofibrils and actomyosin subfragment 1 (acto-S1). At I = 0.2 M, pH 7, and 15 degrees C, the inhibition constants for rabbit myofibrils are 0.17, 3, and 5 mM, respectively; the values for frog myofibrils at 0 degrees C are very similar, being 0.22, 1.5, and 2.5 mM. The inhibition constant of AMPPNP is about 2 orders of magnitude larger than the reported dissociation constant for fibers [Marston, S. B., Rodger, C. D., & Tregear, R. T. (1976) J. Mol. Biol. 104, 263-276]. A possible reason for this difference is that AMPPNP binding results in the dissociation of one head of each myosin molecule. The inhibition constants for rabbit acto-S1 cross-linked with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide measured under the same conditions were 0.12, 2.6, and 3.5 mM for ADP, PPi, and AMPPNP, respectively. The inhibition of cross-linked and native acto-S1 was compared at low ionic strength and was found to be similar. The value for ADP is very similar to reported values of the dissociation constant whereas the inhibition constants for AMPPNP and PPi are an order of magnitude weaker [Greene, L. E., & Eisenberg, E. (1980) J. Biol. Chem. 255, 543-548].

Seven new nontoxinogenic (tox) mutants of corynebacteriophage beta were isolated. Strains of Cornyebacterium diphtheriae C 7 lysogenic for these tox mutants of beta were tested for their ability to produce extracellular diphtherial toxin or proteins (CRMs) that cross-react immunologically with toxin. By using a sensitive reversed passive hemagglutination assay for toxin antigen, three of the tox mutants were phenotypically CRM+ and four were CRM-. The molecular weights of the CRMs produced by mutants beta tox-1, beta tox-2, and beta tox-3 were determined to be approximately 20,000, 26,000, and 34,000, respectively, by electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate. The 26,000 and 34,000-dalton CRMs had nicotinamide adenine dinucleotide: elongation factor 2 adenosine diphosphate ribose transferase activity, but the 20,000-dalton CRM did not. These three CRMs correspond to amino-terminal fragments of diphtherial toxin and appear to be formed by chain termination during protein synthesis directed by phages with nonsense mutations in the structural gene for diphtherial toxin. No complementation was observed between independently isolated tox mutants of phage beta. The positions of four tox markers on the vegetative genetic map of phage beta were determined, and the orientation of transcription of the structural gene for diphtherial toxin with respect to other markers on the genetic map of phage beta was established.

We have used adenosine diphosphate analogs containing electron paramagnetic resonance (EPR) spin moieties and EPR spectroscopy to show that the nucleotide-binding site of kinesin-family motors closes when the motor.diphosphate complex binds to microtubules. Structural analyses demonstrate that a domain movement in the switch 1 region at the nucleotide site, homologous to domain movements in the switch 1 region in the G proteins [heterotrimeric guanine nucleotide-binding proteins], explains the EPR data. The switch movement primes the motor both for the free energy-yielding nucleotide hydrolysis reaction and for subsequent conformational changes that are crucial for the generation of force and directed motion along the microtubule.

The environment near the ribose binding site of skeletal myosin subfragment 1 (S1) was investigated by use of two adenosine 5'-diphosphate analogues with fluorescent groups attached at the 2'- and 3'-hydroxyls of the ribose ring. We have compared steady-state and time-resolved fluorescent properties of the reversibly bound S1-nucleotide complexes and the complexes generated by N,N'-p-phenylenedimaleimide (pPDM) thiol cross-linking or vanadate (Vi) trapping. A new fluorescent probe, 2'(3')-O-[N-[2-[[[5-(dimethylamino)naphthyl]sulfonyl] amino]ethyl]carbamoyl]adenosine 5'-diphosphate (DEDA-ADP), which contains a base-stable carbamoyl linkage between the ribose ring and the fluorescent dansyl group, was synthesized and characterized. For comparison, we performed parallel experiments with 2'(3')-O-(N-methylanthraniloyl)adenosine 5'-diphosphate (MANT-ADP) [Hiratsuka, T. (1983) Biochim. Biophys. Acta 742, 496-508]. Solute quenching studies indicated that both analogues bound reversibly to a single cleft or pocket near the ribose binding site. However, steady-state polarization measurements indicated that the probes were not rigidly bound to the protein. The quantum yields of both fluorophores were higher for the complexes formed after trapping with pPDM or Vi than for the reversibly bound complexes. Both DEDA-ADP and MANT-ADP, respectively, had nearly homogeneous lifetimes free in solution (3.65 and 4.65 ns), reversibly bound to S1 (12.8 and 8.6 ns), and trapped on S1 by pPDM (12.7 and 8.7 ns) or Vi (12.8 and 8.6 ns). In contrast to the quantum yields, the lifetimes were not increased upon trapping, compared to those of the reversibly bound states. These results suggested that static quenching in the reversibly bound complex was relieved upon trapping. Taken together, the results suggest that there was a conformational change near the ribose binding site upon trapping by either pPDM or Vi. On the basis of the quantum yield, lifetime, polarization, and solute accessibility studies, we could not detect differences between the S1-pPDM-nucleotide analog complex and the S1-Vi-nucleotide analogue complex for either analogue. Thus, previously observed differences with the adenine modified nucleotide analogue 1,N6-ethenoadenosine diphosphate (epsilon ADP) could not be detected with these ribose-modified probes, indicating that structural differences may be localized to the adenine binding site and not transmitted to the region near the ribose ring.

Enteric inhibitory neurotransmission is an important feature of the neural regulation of gastrointestinal motility. Purinergic neurotransmission, via P2Y1 receptors, mediates one phase of inhibitory neural control. For decades, ATP has been assumed to be the purinergic neurotransmitter and smooth muscle cells (SMCs) have been considered the primary targets for inhibitory neurotransmission. Recent experiments have cast doubt on both of these assumptions and suggested that another cell type, platelet-derived growth factor receptor-α-positive (PDGFRα(+)) cells, is the target for purinergic neurotransmission. We compared responses of PDGFRα(+) cells and SMCs to several purine compounds to determine if these cells responded in a manner consistent with enteric inhibitory neurotransmission. ATP hyperpolarized PDGFRα(+) cells but depolarized SMCs. Only part of the ATP response in PDGFRα(+) cells was blocked by MRS 2500, a P2Y1 antagonist. ADP, MRS 2365, β-NAD, and adenosine 5-diphosphate-ribose, P2Y1 agonists, hyperpolarized PDGFRα(+) cells, and these responses were blocked by MRS 2500. Adenosine 5-diphosphate-ribose was more potent in eliciting hyperpolarization responses than β-NAD. P2Y1 agonists failed to elicit responses in SMCs. Small hyperpolarization responses were elicited in SMCs by a small-conductance Ca(2+)-activated K(+) channel agonist, cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine, consistent with the low expression and current density of small-conductance Ca(2+)-activated K(+) channels in these cells. Large-amplitude hyperpolarization responses, elicited in PDGFRα(+) cells, but not SMCs, by P2Y1 agonists are consistent with the generation of inhibitory junction potentials in intact muscles in response to purinergic neurotransmission. The responses of PDGFRα(+) cells and SMCs to purines suggest that SMCs are unlikely targets for purinergic neurotransmission in colonic muscles.

Cyclic adenosine diphosphate-ribose, an endogenous metabolite of nicotinamide adenine dinucleotide was first characterized as a potent Ca2+ mobilizing agent in sea urchin eggs. Mounting evidence points to it being an endogenous activator of Ca(2+)-induced Ca2+ release by non-skeletal muscle ryanodine receptors in several invertebrate and mammalian cell types. Cyclic adenosine diphosphate-ribose is synthesized by adenosine diphosphate-ribosyl cyclases, which have been found to be widespread enzymes. Recent data suggests that cyclic adenosine diphosphate-ribose may function as a second messenger in sea urchin eggs at fertilization and in stimulus secretion coupling in pancreatic beta-cells. A second messenger role for cyclic adenosine diphosphate-ribose requires that its intracellular levels be under the control of extracellular stimuli. Another second messenger, cGMP, stimulates the synthesis of cyclic adenosine diphosphate-ribose from nicotinamide adenine dinucleotide by activating the adenosine diphosphate-ribosyl cyclase pathway in sera urchin eggs and egg homogenates, suggesting that cyclic adenosine diphosphate-ribose may be an intracellular messenger for cell surface receptors or nitric oxide, which activate cGMP-producing guanylate cyclases. Cyclic adenosine diphosphate-ribose may have a similar role to inositol trisphosphate in controlling intracellular calcium signalling with these two calcium-mobilizing second messengers activating ryanodine receptors and inositol trisphosphate receptors respectively.

DNA repair is essential for the survival of both normal and cancer cells. An elaborate set of signaling pathways detect single-strand and double-strand DNA breaks and mediate either DNA repair or apoptosis if the damage is too great to repair. Poly(adenosine diphosphate [ADP]-ribose) polymerases (PARPs) play a key role in the repair of base damage via the base excision repair pathway. Pharmacological inhibition of PARP induces cell death in tumors with mutations in certain DNA repair pathways--such as the BRCA pathways of double-strand break repair--and when combined with chemotherapies that cause DNA damage. PARP inhibitors are being investigated as a monotherapy for the treatment of patients with BRCA 1/2 mutations; in the treatment of triple-negative breast cancer, because of its molecular similarities to BRCA1-mutated malignancies; and as a strategy to potentiate the DNA-damaging effects of chemotherapy and radiation. The aim of this article is to review the preclinical data and rationale for PARP inhibitor use in the aforementioned settings, as well as the current status of the clinical development of these agents in the treatment of breast cancer, along with future directions for research in this field. Trials have been identified via searches of PubMed, clinicaltrials.gov, and the Proceedings of the American Society of Clinical Oncology Annual Meeting and the San Antonio Breast Cancer Symposium.

Following stimulation with adenosine diphosphate (ADP), collagen, or arachidonic acid, unstirred human platelet suspensions bind 125I-fibrinogen in a reaction that reaches completion within 30 min. Scatchard analysis of these binding data reveals two sets of binding sites with all 3 agents: a high affinity site (Kd 0.029-0.045 microM) binding 1000-1600 fibrinogen molecules per platelet, and a lower affinity site (Kd 1.2-2.0 microM) binding 46,000-76,000 fibrinogen molecules per platelet. At a concentration of apyrase that inhibited ADP-induced fibrinogen binding by greater than 85%, fibrinogen binding induced by collagen and arachidonic acid was only partially affected. This suggests that fibrinogen binding induced by collagen or arachidonic acid does not require released ADP. We isolated a monoclonal antibody, B59.2, which precipitated the glycoprotein IIb-IIIa complex from solubilized platelet membranes. Binding of labeled antibody to platelets before or after exposure to ADP, collagen, or arachidonic acid showed a single class of approximately 22,000 binding sites with Kd 0.019 microM. Binding of B59.2 was complete within 1 min and was not inhibited by EDTA. Preincubation of platelet suspensions with a 2.1 microM concentration of B59.2 caused inhibition of secretion and aggregation, but not of thromboxane-B2 synthesis, in response to 1 microgram/ml collagen, 40 microM arachidonic acid, or 4 microM ADP, concentrations of aggregating agents that produced complete aggregation and secretion in the absence of B59.2. At this concentration of B59.2, fibrinogen binding to stimulated platelets was inhibited by approximately 45%-55%. These data demonstrate that collagen and arachidonic acid can expose fibrinogen binding sites independently of released ADP; and that the glycoprotein IIb-IIIa complex is involved in secretion, aggregation, and fibrinogen binding, but not in thromboxane synthesis occurring in response to collagen, arachidonic acid, or ADP.

Type 4 cyclic adenosine monophosphate (cAMP) phosphodiesterase (PDE4) inhibitors and other agents that raise intracellular cAMP levels induce apoptosis in B-cell chronic lymphocytic leukemia (B-CLL) but not in T-CLL or peripheral blood T cells. Two principal effector proteins for cAMP are protein kinase A (PKA) and EPAC (exchange protein directly activated by cAMP), a Rap guanosine 5'-diphosphate (GDP) exchange factor. We here examine whether varying expression of EPAC accounts for the discrepant sensitivity of B-CLL and T cells to PDE4 inhibitor-induced apoptosis. B-CLL and peripheral blood B cells express EPAC1 transcript, whereas T-CLL, peripheral blood T cells, monocytes, and neutrophils do not. Treatment with the PDE4 inhibitor rolipram induces Rap1 activation in B-CLL cells but not in peripheral blood B cells, T-CLL, or any of the normal hematopoietic lineages examined. The EPAC-specific cAMP analog 8CPT-2Me-cAMP (8-(4-chloro-phenylthio)-2'-O-methyladenosine-3',5'-cAMP) activates Rap1 in B-CLL cells, but, unlike rolipram/forskolin or 8-Bromo-cAMP, it does not induce PKA activation, as judged by phosphorylation of the transcription factor cAMP-response element binding protein (CREB). Unexpectedly, whereas rolipram/forskolin and 8-Bromo-cAMP induce apoptosis in B-CLL cells, 8CPT-2Me-cAMP decreased basal apoptosis in B-CLL cells by an average of 25% (P<.002). Our results demonstrate that B-CLL cells uniquely activate Rap1 in response to PDE4 inhibitors and suggest that physiologic stimuli that activate EPAC may transmit an antiapoptotic signal.

Pertussis toxin (PT) is a known mitogen for T lymphocytes. The mechanism by which the toxin stimulates proliferation has remained obscure and paradoxical because, in some types of cells, the toxin also inhibits growth factor-mediated signal transduction. It has previously been shown that the adenosine-diphosphate ribosyltransferase activity of the toxin is not required to produce the mitogenic effect. A biochemical explanation for the mitogenic activity has therefore remained obscure. We investigated the biochemical basis for the mitogenic activity of PT by using the transformed human T cell line, Jurkat. PT stimulated a rapid rise in cytosolic-free [Ca2+] from both intra- and extracellular sources. This was associated with an increase in the cellular diacylglycerol and inositol triphosphate levels with a concomitant decrease in the levels of phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate. The half-maximal effective dose of PT was 1.7 nM. PT also stimulated the production of interleukin 2. Only the holotoxin or B-oligomer (the presumptive membrane-binding subunit) was capable of stimulating an increase in [Ca2+] in these cells. This activity of PT mimicked that of some anti-T3-T cell antigen receptor complex monoclonal antibodies that also stimulate increases in the second messengers, diacylglycerol and Ca2+. The effects of PT and anti-T3 complex antibody were identical and not additive in Jurkat cells, suggesting that both agents were activating the same signal transduction pathway. These data provide a mechanistic explanation for the mitogenic effects of PT and suggest that the toxin may be interacting with a specific receptor in the T lymphocyte plasma membrane.

BACKGROUND

Heart muscle primarily relies on adenosine triphosphate produced by oxidative phosphorylation and is highly vulnerable to anoxic insult. Although a number of strategies aimed at improving myopreservation are available, no effective means of preserving mitochondrial energetics under conditions of anoxic injury have been developed. Openers of mitochondrial adenosine triphosphate-sensitive potassium channels have emerged as powerful cardioprotective agents presumably capable of maintaining mitochondrial function under metabolic stress. Here, we evaluated the ability of a prototype mitochondrial adenosine triphosphate-sensitive potassium channel opener, diazoxide, to preserve oxidative phosphorylation in mitochondria subjected to anoxia and reoxygenation.

METHODS

Mitochondria were isolated from rat hearts and subjected to 20 minutes of anoxia, followed by reoxygenation. Mitochondrial respiration and oxidative phosphorylation, as well as mitochondrial integrity, were assessed by means of ion-selective minielectrodes, high-performance liquid chromatography, fluorometry, and electron microscopy.

RESULTS

Anoxia-reoxygenation decreased the rate of adenosine diphosphate-stimulated oxygen consumption, inhibited adenosine triphosphate production, and disrupted mitochondrial integrity. On average, anoxic stress reduced adenosine diphosphate-stimulated respiration from 291 +/- 14 to 141 +/- 15 ng-atoms O(2). min(-1). mg(-1) protein and decreased the rate of adenosine triphosphate production from 752 +/- 14 to 414 +/- 34 nmol adenosine triphosphate. min(-1). mg(-1) protein. After anoxia, the majority (88%) of mitochondria was damaged or swollen and released adenylate kinase, a marker of mitochondrial integrity. Diazoxide (100 micromol/L), present throughout anoxia, preserved adenosine diphosphate-stimulated respiration at 255 +/- 7 ng-atoms O(2). min(-1). mg(-1) protein and adenosine triphosphate production at 640 +/- 39 nmol adenosine triphosphate. min(-1). mg(-1) protein. Diazoxide also protected mitochondrial structure from anoxia-mediated damage, so that after anoxic stress, 67% of mitochondria remained intact and adenylate kinase was confined to the mitochondria.

CONCLUSIONS

The present study demonstrates that diazoxide diminishes anoxia-induced functional and structural deterioration of cardiac mitochondria. By protecting mitochondria and preserving myocardial energetics, diazoxide may be useful under conditions of reduced oxygen availability, including global surgical ischemia or storage of donor heart.

Increased ecto-5'-nucleotidase (ecto-5'NT) protein expression in several multidrug-resistant (MDR) cell lines, documented previously by our group, suggests that this enzyme is involved in drug resistance. Here, Northern blot analysis of selected cell lines and their MDR variants positively correlated ecto-5'NT protein with its mRNA expression. An inhibitor of ecto-5'NT enzymatic activity, alpha,beta-methyleneadenosine 5'-diphosphate (AMP-CP), was used to determine if functionally active enzyme had a role in drug resistance. AMP-CP (0.3 mM) reversed the resistance of ecto-5'NT-positive MDR cells (MCF7/A6, L1210/A) to doxorubicin, whereas it did not affect the doxorubicin sensitivity of the ecto-5'NT-negative parental cell lines or that of 2 ecto-5'NT-negative MDR cell lines (HL60/VCR and A2780/DX5). Furthermore, AMP-CP increased rhodamine uptake and inhibited rhodamine efflux from ecto-5'NT-positive MDR cells without affecting ecto-5'NT-negative MDR cells. The presence of exogenous adenosine (0.5 microM) circumvented AMP-CP-induced inhibition of rhodamine efflux from EL4/ADM cells. AMP-CP inhibited the growth of the ecto-5'NT-positive L1210/A MDR cells but had no effect on the growth of the parental cell line. Determination of intracellular ATP levels indicated that MDR cells which had increased ecto-5'NT expression also had a lower intracellular ATP level than their parental cells. Our results suggest that, in certain MDR cell lines, ecto-5'NT serves as a required accessory molecule in resistance mediated by ATP-dependent mechanisms and that growth-sustaining nucleosides are provided by this salvage pathway.

The criterion for chemical equilibrium at specified temperature, pressure, pH, concentration of free magnesium ion, and ionic strength is the transformed Gibbs energy, which can be calculated from the Gibbs energy. The apparent equilibrium constant (written in terms of the total concentrations of reactants like adenosine 5'-triphosphate, rather than in terms of species) yields the standard transformed Gibbs energy of reaction, and the effect of temperature on the apparent equilibrium constant at specified pressure, pH, concentration of free magnesium ion, and ionic strength yields the standard transformed enthalpy of reaction. From the apparent equilibrium constants and standard transformed enthalpies of reaction that have been measured in the adenosine 5'-triphosphate series and the dissociation constants of the weak acids and magnesium complexes involved, it is possible to calculate standard Gibbs energies of formation and standard enthalpies of formation of the species involved at zero ionic strength. This requires the convention that the standard Gibbs energy of formation and standard enthalpy of formation for adenosine in dilute aqueous solutions be set equal to zero. On the basis of this convention, standard transformed Gibbs energies of formation and standard transformed enthalpies of formation of adenosine 5'-trisphosphate, adenosine 5'-diphosphate, adenosine 5'-monophosphate, and adenosine at 298.15 K, 1 bar, pH = 7, a concentration of free magnesium ions of 10(-3) M, and an ionic strength of 0.25 M have been calculated.