Abamectin causes cardiac dysfunction in carp via inhibiting redox equilibrium and resulting in immune inflammatory response and programmed cell death.

This study aims to investigate the effects of environmentally relevant concentrations of abamectin on the cardiac function of carp and the potential mechanisms. Here, male carp were exposed to abamectin, and cardiac function-related enzymatic markers were examined. Cardiac histopathology, redox equilibrium, inflammation, and cell death were evaluated. Abamectin exposure caused cardiac dysfunction by upregulating lactate dehydrogenase (LDH), aspartate aminotransferase (AST), creatine kinase (CK), creatine Kinase MB isoenzyme (CK-MB) and white blood cells (WBCs), and decreasing red blood cells (RBCs) and hemoglobin (Hb). DHE staining and biochemical assays revealed that abamectin caused ROS release and oxidative stress by inhibiting Nrf2-ARE pathway. Histopathological and real-time fluorescence quantitative PCR (RT-qPCR) assays revealed that abamectin caused myocardial fiber swelling and inflammatory cell infiltration, enhanced pro-inflammatory cytokines tumor necrosis factor-α (Tnf-α), interleukin-1 beta (Il-1ß), and Il-6 levels and attenuated anti-inflammatory cytokines Il-10 and transforming growth factor beta 1 (Tgf-ß1) through activating NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome and nuclear factor kappa-B (NF-κB) pathway. Tunel staining showed that abamectin triggered cardiac apoptosis via activating p53-mediated mitochondrial apoptosis with elevated bcl2-associated X (Bax), reduced B-cell lymphoma-2 (Bcl-2), and activated Caspase-9 and Caspase-3. Immunoblot analysis revealed that abamectin activated autophagic flow by inhibiting mammalian target of rapamycin (mTOR), resulting in the conversion of LC3B from LC3-I to LC3-II, elevation of autophagy protein 5 (Atg5), and reduction of p62. Overall, abamectin caused cardiac dysfunction in carp via inhibiting redox equilibrium and resulting in immune inflammatory response and programmed cell death.

Shenlian extract attenuates myocardial ischaemia-reperfusion injury via inhibiting M1 macrophage polarization by silencing miR-155.

CONTEXT: Shenlian extract (SL) is a combination of Salvia miltiorrhiza Bge. (Labiatae) and Andrographis paniculata (Burm. F.) Wall. Ex Nees (Acanthaceae) extracts, which promote blood circulation and clear endogenous heat toxins. Myocardial ischaemia-reperfusion injury (MI/RI) is aggravated myocardial tissue damage induced by reperfusion therapy after myocardial infarction. OBJECTIVES: This study explores the effect of SL on MI/RI and the underlying mechanism. MATERIALS AND METHODS: Primary peritoneal macrophages (pMACs) were treated with LPS and SL (5, 10 or 20 µg/mL) for 24 h. The myocardial ischaemia-reperfusion (MI/R) model was established after administration of different doses of SL (90, 180 or 360 mg/kg). Myocardial tissue injury was assessed by methylthiazolyl tetrazolium (TTC) staining and levels of creatine kinase (CK), lactate dehydrogenase (LDH) and superoxide dismutase (SOD) in mice. The double immunofluorescence staining of iNOS/F4/80 and CD86/F4/80 was used to detect macrophage M1 polarization. The levels of miR-155, inflammatory factors and chemokines were detected by qRT-PCR or ELISA. CD86, iNOS, SOCS3, JAK2, p-JAK2, STAT3 and p-STAT3 proteins expressions in macrophages were analyzed by western blotting. Conditioned medium transfer systems were designed to unite M1 macrophages with H/R cardiomyocytes, and cell apoptosis was detected by TUNEL staining, western blotting or immunohistochemistry. RESULTS: SL reduced apoptosis, diminished CK and LDH levels, raised SOD concentration and decreased infarct size in the MI/R model. Meanwhile, SL decreased miR-155 level, inhibited M1 macrophage polarization and inflammation. Furthermore, SL promoted SOCS3 expression and blocked JAK2/STAT3 pathway in vitro. CONCLUSIONS: SL may be a promising TCM candidate for MI/RI. The underlying mechanisms could be associated with inhibition of M1 macrophage polarization via down-regulating miR-155.

Ameliorative effect of flavocoxid on cyclophosphamide-induced cardio and neurotoxicity via targeting the GM-CSF/NF-κB signaling pathway.

Cyclophosphamide (Cyclo) is a chemotherapeutic agent used as an immunosuppressant and as a treatment for many cancerous diseases. Many previous pieces of literature proved the marked cardio and neurotoxicity of the drug. Thus, this research provides evidence on the alleviative effect of flavocoxid on the cardiac and brain toxicity of cyclophosphamide in mice and determines its underlying mechanisms. Flavocoxid (Flavo) is a potent antioxidant and anti-inflammatory agent that inhibits the peroxidase activity of cyclooxygenase (COX-1 and COX-2) enzymes and 5-lipooxygenase (5-LOX). Flavo was administered orally (20 mg/kg) for 2 weeks, followed by Cyclo (100 mg/kg, i.p.) on day 14. Higher heart and brain weight indices, serum lactate dehydrogenase (LDH), creatine kinase (CK-MB), and nitric oxide (NO) were mitigated following Flavo administration. Flavo modulated oxidative stress biomarkers (malonaldehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD)), tumor necrosis factor-α (TNF-α), and interleukin (IL)-1ß. Additionally, cardiac troponin I (cTn-I), nuclear factor kappa B (NF-κB), brain amyloid precursor protein (APP), and granulocyte macrophage colony-stimulating factor (GM-CSF) were decreased by Flavo administration. Moreover, Flavo ameliorated heart and brain histopathological changes and caspase-3 levels. Collectively, Flavo (20 mg/kg) for 14 days showed significant cardio and neuroprotective effects due to its antioxidant, anti-inflammatory, and antiapoptotic activities via modulation of oxidative stress, inflammation, and the GM-CSF/NF-κB signaling pathway.

Ginkgolide B targets and inhibits creatine kinase B to regulate the CCT/TRiC-SK1 axis and exerts pro-angiogenic activity in middle cerebral artery occlusion mice.

Promoting angiogenesis in the ischemic penumbra is a well-established method of ischemic stroke treatment. Ginkgolide B (GB) has long been recognized for its neuroprotective properties following stroke. As previously reported, it appears that stroke-induced neurogenesis and angiogenesis interact or are dependent on one another. Although the pharmacodynamic effect of GB on cerebral blood flow (CBF) following ischemic stroke has been reported, the molecular mechanism underlying this effect remains unknown. As such, this study sought to elucidate the pharmacodynamic effects and underlying mechanisms of GB on post-stroke angiogenesis. To begin, GB significantly increased the proliferation, migration, and tube formation capacity of mouse cerebral hemangioendothelioma cells (b.End3) and human umbilical vein endothelial cells (HUVEC). Additionally, GB significantly improved angiogenesis after oxygen-glucose deprivation/reperfusion (OGD/R) in endothelial cells. The dynamics of CBF, brain microvascular neovascularization and reconstruction, and brain endothelial tissue integrity were examined in middle cerebral artery occlusion (MCAO) mice following GB administration. Through label-free target detection techniques, we discovered for the first time that GB can specifically target Creatine Kinase B (CKB) and inhibit its enzymatic activity. Additionally, we demonstrated through network pharmacology and a series of molecular biology experiments that GB inhibited CKB and then promoted angiogenesis via the CCT/TRiC-SK1 axis. These findings shed new light on novel therapeutic strategies for neurological recovery and endothelial repair following ischemic stroke using GB therapy.

D-limonene (5 (one-methyl-four-[1-methylethenyl]) cyclohexane) diminishes CCl4-induced cardiac toxicity by alleviating oxidative stress, inflammatory and cardiac markers.

Background: The cardiovascular crisis is advancing rapidly throughout the world. A large number of studies have shown that plant polyphenols affect major mechanisms involved in cardiovascular events through their action on the antioxidant system, signaling, and transcription pathways. D-limonene, a monocyclic monoterpene obtained from citrus fruits, is reported to possess many pharmacological activities.Methods: The experiment was designed to determine the protective effect of D-limonene against cardiac injury induced by CCl4 in Wistar rats. Rats were treated with two doses of D-limonene against cardiac injury induced by CCl4. Serum toxicity markers, cardiac toxicity biomarker enzymes, inflammatory mediators, anti-oxidant armory, lipid peroxidation, lipid profile, and histology were done.Results: CCl4 intoxication resulted in a substantial rise in FFA, TC, TG, PL, LDL, VLDL, and a reduction in HDL, restoring these changes with the administration of D-limonene at a dosage of 200 mg/kg. CCl4 administration also resulted in lipid oxidation and decreased antioxidant activity. At the same time, D-limonene at a dosage of 200 mg/kg body weight inhibited LPO and restored in vivo antioxidant components to normal. CCl4 intoxication also resulted in a significant increase in inflammatory markers like IL-6, TNF-α, high sensitivity Corticotropin Releasing Factor (Hs-CRF), and biomarkers of cardiac toxicity like alanine aminotransferase (ALT), lactate dehydrogenase (LDH), creatine kinase (CK), creatine kinase MB (CKMB), and Troponin I & troponin-t activities. D-limonene reversed all these changes to normal. Histology further confirmed our obtained results.Conclusion: These findings indicate that D-limonene can ameliorate cardiac injury at a 200 mg/kg body weight dosage. Henceforth, D-Limonene intervenes in mediating CCl4 induced toxicity by various signaling pathways.

Selected root plant supplementation reduces indices of exercise-induced muscle damage: A systematic review and meta-analysis.

This systematic review and meta-analysis examined the effects of selected root plants (curcumin, ginseng, ginger and garlic) on markers of muscle damage and muscular performance measures following muscle-damaging protocols. We included 25 studies (parallel and crossover design) with 353 participants and used the PEDro scale to appraise each study. Forest plots were generated to report on standardised mean differences (SMD) and p-values at 24 and 48 hours following the muscle-damaging protocols. The meta-analysis showed that the supplemental (SUPP) condition showed significantly lower levels of indirect muscle damage markers (creatine kinase, lactate dehydrogenase and myoglobin) and muscle soreness at 24 hours and 48 hours (p < 0.01) than the placebo (PLA) condition. The inflammatory markers were significantly lower for the SUPP condition than the PLA condition at 24 hours (p = 0.02), although no differences were identified at 48 hours (p = 0.40). There were no significant differences in muscular performance measures between the SUPP and PLA conditions at 24 hours and 48 hours (p > 0.05) post-exercise. According to our qualitative data, a number of studies reported a reduction in oxidative stress (e.g., malondialdehyde, superoxide dismutase) with a concomitant upregulation of anti-oxidant status, although other studies showed no effects. Accordingly, selected root plants minimised the level of several biomarkers of muscle damage, inflammation and muscle soreness during periods of exercise-induced muscle damage. However, the benefits of these supplements in ameliorating oxidative stress, increasing anti-oxidant status and accelerating recovery of muscular performance appears equivocal, warranting further research in these outcome measures.

The protective effect of chrysin against oxidative stress and organ toxicity in rats exposed to propetamphos.

The aim of this study was to investigate the protective efficacy of chrysin against propetamphos exposure. For this purpose, 2 to 3-month-old 40 male Wistar Albino rats were used. These animals were randomly assigned to four groups. The animals in the control group received the vehicle substance (corn oil) alone. Groups 2, 3 and 4 were administered with 50 mg/kg.bw/day of chrysin (in corn oil), 10 mg/kg.bw/day of propetamphos (in corn oil), and 10 mg/kg.bw/day of propetamphos plus 50 mg/kg.bw/day of chrysin, respectively, for 28 days. Some oxidative stress/lipid peroxidation parameters (MDA, SOD, CAT, GSH-Px, NO, glutathione) and serum biochemical parameters (triglyceride, cholesterol, creatinine, BUN, creatine phosphokinase, ALT, ALP and pseudocholinesterase) were analyzed in tissue/blood samples. Also, histopathological findings were observed. According to the data obtained, no significant alteration had occurred in these parameters and the histological findings in the group given chrysin alone, when compared to the control group. Significant unfavorable alterations were detected in the oxidative stress/lipid peroxidation/antioxidant status parameters, all biochemical parameters and histopathological findings of the group that received propetamphos alone. In the group that was given both chrysin and propetamphos, remedial/recovery alterations were observed in the oxidative stress/lipid peroxidation/antioxidant status values, serum biochemical parameters and histopathological findings, such that the values and histopathological findings showed partly similarity to those of the control group. In result, it is suggested that chrysin may provide protection against propetamphos exposure and propetamphos-induced organ damage in rats at a certain level.

Downhill running in rats: influence on neutrophils, macrophages, and MyoD+ cells in skeletal muscle.

The accumulation of neutrophils and macrophages, as well as the activation of satellite cells, are early events following skeletal muscle injury. We examined the temporal relationship between changes in neutrophils, macrophages, and MyoD protein, a marker of satellite cell activation, after injurious exercise. Male rats ( n=47) performed an intermittent downhill (-16% grade) running (17 m/min) protocol and the solei were obtained at 0, 2, 6, 24, 48, or 72 h post-exercise. Neutrophils, macrophages (ED1 and ED2), and MyoD+ cells were determined in muscle cross sections using immunohistochemistry. Downhill running increased ( P

Plasma creatine kinase activity and glutathione after eccentric exercise.

PURPOSE: This study examined whether plasma total glutathione levels could explain the intersubject variability in the creatine kinase (CK) response to eccentric exercise. We hypothesized that the increase in plasma CK activity after eccentric exercise would be lower for individuals with low plasma total glutathione (<2.5 micromol x L-1) compared with individuals with high total glutathione (>3.8 micromol x L-1), but other indicators of muscle damage would be the same between groups. METHODS: Resting blood samples were obtained over 2 d from 60 subjects and analyzed for plasma total glutathione. Eight subjects who had total glutathione values below 2.5 micromol x L-1 (LG), and nine who had values above 3.8 micromol x L-1 (HG) performed 50 maximal eccentric actions of the elbow flexors. Maximal voluntary isometric contraction (MVC), relaxed arm angle (RANG), and blood samples for CK, myoglobin (Mb), and total glutathione were obtained pre, post (except blood samples), 24, 48, 72, 96, and 120 h after exercise. RESULTS: There was a significant group-by-time interaction in analysis of MVC, RANG, total glutathione, CK, and Mb response to exercise. Although LG showed a smaller CK response to eccentric exercise compared with HG, LG also showed a smaller increase in plasma Mb, a faster recovery of MVC and RANG, and an increase in plasma total glutathione. CONCLUSION: Subjects with low plasma total glutathione levels had a smaller plasma CK and Mb response and a faster recovery from eccentric exercise compared with subjects having high plasma total glutathione levels. We suggest that a blunted inflammatory response in subjects with low plasma glutathione may be one explanation for these findings.

Necrotising myopathy, an unusual presentation of a steroid-responsive myopathy.

OBJECTIVE: To evaluate the clinical features, muscle pathology and response to treatment in patients with a necrotising myopathy, without mononuclear cell infiltrates. BACKGROUND: Mononuclear cell infiltrates in the muscle biopsy specimen are the diagnostic hallmark of the immune-mediated idiopathic inflammatory myopathies (IIM). In patients with the typical clinical features of IIM, absence of these infiltrates in the muscle biopsy specimen casts doubt on the diagnosis and leads to uncertainty about therapeutical strategies. METHODS: A detailed description is given of the clinical, laboratory, and histopathological features of eight patients suspected of having an idiopathic inflammatory myopathy, in whom mononuclear cell infiltrates in their muscle biopsy specimens were lacking. RESULTS: Eight patients (five men, three women, age range 40-69 years) had severe, symmetrical proximal weakness with a subacute onset. There were no skin abnormalities suggesting dermatomyositis. Serum creatine kinase activity was more than 10 times elevated. Repeated muscle biopsy specimens, taken from a symptomatic muscle prior to immunosuppressive treatment showed widespread necrosis, regeneration, and atrophy of muscle fibres, but no mononuclear cell infiltrates. Known causes of necrotising myopathy were excluded. Three patients had a malignancy. Adequately dosed and sustained immunosuppressive treatment eventually resulted in normal or near normal muscle strength in seven patients. One patient showed marked improvement. CONCLUSION: Occasionally, patients who clinically present as an idiopathic inflammatory myopathy may lack mononuclear cell infiltrates in their muscle biopsy specimens. This subacute-onset progressive necrotising myopathy should not deter the clinician from timely and appropriate treatment as we consider this myopathy to be steroid-responsive with a possible immune-mediated pathogenesis.

Inhibition of the yeast V-type ATPase by cytosolic ADP.

The activity of the vacuolar H(+)-ATPase has been characterized in isolated vacuoles of the yeast Saccharomyces cerevisiae by means of the patch-clamp technique. With cytosolic calcium at virtually zero (<10(-9) M), Mg-ATP induced a transient, bafilomycin A(1)-sensitive current corresponding to the flow of positive charges from the cytoplasmic surface to the vacuolar lumen. The Mg-ATP-dependent current reached its maximum amplitude (30+/-8 mA m(-2) with 5 mM Mg-ATP, n=34) within 15-20 s and declined slowly over a period of about 15-20 min even in the continuous presence of Mg-ATP. This decline of pumping activity was independent of the cytosolic KCl concentration, suggesting an inhibitory mechanism different from the high salt-induced dissociation of V(0) and V(1) reported for the V-ATPase of plants and fungi. Cytosolic ADP was found to modulate the pump activity since Mg-ATP-induced pump current was smaller if monitored in the presence of 5 mM ADP and addition of 5 mM ADP in the presence of 5 mM Mg-ATP reduced the pump current by more than 50%. Furthermore, reduction of the cytosolic ADP concentration by the ATP-regenerating system creatine phosphate/creatine kinase partially relieved the endogenous inhibition of the V-ATPase, confirming that interaction of cytosolic ADP with the V-ATPase is the reason for the transient nature of the pump current in yeast vacuoles.

The central role of thromboxane and platelet activating factor receptors in ex vivo regulation of endotoxin-induced monocyte tissue factor activity in human whole blood.

Expression of tissue factor (TF) by activated monocytes may initiate thrombotic episodes associated with diseases, such as thrombosis and atherosclerosis. In this study, steps in the regulatory pathways of lipopolysaccharide (LPS)-induced monocyte TF activity and released TNF-alpha in human whole blood were probed for using an array of inhibitors, comprising specific inhibitors of cytosolic phospholipase A(2) (PLA(2)) (AACOCF(3)), secretory PLA(2) (SB-203347), protein kinase (PK) (staurosporine), PKC (GF-109203; BIM), and serine protease (Pefabloc SC), antagonists of thromboxane prostanoid (TP) receptor (R) (SQ-29548), platelet activating factor (PAF) R (BN-52021), leukotriene B(4) R (SC-41930), serotonin R (cyproheptadine), fibronectin/fibrinogen R (RGDS), and finally, creatine phosphate/creatine phosphokinase (CP/CPK) which removes ADP. Whereas when added alone neither of these agents significantly inhibited LPS-induced TF or TNF-alpha, when presented as a reference cocktail comprising all the agents, TF activity and TNF-alpha were reduced by 77% and 49%, respectively. By subsequently testing a series of incomplete inhibitory cocktails equal to the reference except for deleted single agents or combinations of two or three active agents, the inhibitory effect of the reference cocktail could be shown to depend on the presence of the protease inhibitor and the thromboxane A(2) and PAF antagonists.

Exogenous neopterin causes cardiac contractile dysfunction in the isolated perfused rat heart.

Neopterin is known in humans as a sensitive marker for diseases associated with increased activity of the cellular immune system. Recent studies report neopterin also to exhibit distinct effects: neopterin induces inducible nitric oxide synthase expression in rat vascular smooth muscle cells and activates translocation of nuclear factor- kappa B. Neopterin may also induce oxidative stress causing apoptotic cell death, or superinduce tumor necrosis factor- alpha -mediated apoptosis. Observing these effects in cell cultures, we were interested in possible consequences of neopterin on cardiac function in the isolated perfused rat heart. The influence of neopterin in three different concentrations (10 micromol/l, 50 micromol/l, 100 micromol/l) on cardiac contractility parameters and coronary vascular resistance were studied in 67 male Sprague-Dawley rats using the temperature-controlled and pressure-constant Langendorff apparatus with retrograde perfusion of the aorta with a Krebs-Henseleit buffer. Treatment with 100 micromol/l neopterin resulted in a significant decrease in coronary flow and cardiac contractility. Coronary flow decreased from 15.2 to 9.5 ml/min (P=0.002), left ventricular pressure from 80 to 52 mmHg (P=0. 002), rate of pressure fall from 1605 to 923 mmHg/s (P=0.001) and rate of pressure rise from 2862 to 1709 mmHg/s (P=0.001). Concentrations lower than 100 micromol/l neopterin had no significant effect on cardiac function. Our study demonstrates a considerable influence of exogenous neopterin on cardiac performance in the Langendorff model of isolated perfused rat hearts. This has to be considered a potential pathogenic factor of cardiac disturbances in diseases in which high concentrations of neopterin are released due to immune activation. At present the exact mechanism remains unclear.

Preservation of mitochondrial function by diazoxide during sustained ischaemia in the rat heart.

1. A possible mechanism for the action of the K(ATP) channel opener diazoxide on the improvement of energy metabolism of ischaemic/reperfused hearts was examined. 2. Isolated, perfused rat hearts were subjected to 40 min ischaemia followed by 60 min reperfusion. Diazoxide at concentrations of 3 to 30 microM was present in the perfusion buffer for the last 15 min of pre-ischaemia. 3. Treatment of the perfused heart with diazoxide enhanced the post-ischaemic recovery of rate-pressure product, attenuated the post-ischaemic rise in left ventricular end-diastolic pressure, and suppressed the release of creatine kinase and purine nucleosides and bases from the reperfused heart. Treatment of the heart with diazoxide also restored myocardial ATP and creatine phosphate and attenuated the decrease in mitochondrial oxygen consumption rate after reperfusion. This attenuation was maintained at the end of ischaemia as well as at the end of reperfusion. 4. In another set of experiments, myocardial skinned bundles were incubated for 30 min under hypoxic conditions in the presence and absence of diazoxide, and then the mitochondrial oxygen consumption rate was determined. Hypoxia induced a decrease in the mitochondrial oxygen consumption rate of the skinned bundles to approximately 40% of the pre-hypoxic value. In contrast, treatment of the bundles with 30 microM diazoxide preserved the normal mitochondrial oxygen consumption rate during hypoxia. This effect was abolished concentration-dependently by the combined treatment with either the K(ATP) channel blocker glibenclamide or 5-hydroxydecanoate. 5. These results suggest that diazoxide is capable of attenuating ischaemia/reperfusion injury of isolated perfused hearts due to preservation of mitochondrial function during ischaemia.

Formation of PI 3-kinase products in platelets by thrombin, but not collagen, is dependent on synergistic autocrine stimulation, particularly through secreted ADP.

Platelet activation by thrombin or collagen results in secretion and synthesis of several platelet agonists that enhance the responses to the primary agonists (autocrine stimulation). To disclose the effects of thrombin and collagen on the phosphorylation of 3-phosphoinositides per se we incubated platelets with five inhibitors of platelet autocrine stimulation (IAS) that act extracellularly. We found that IAS almost totally blocked thrombin-induced production of phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P(2)] and phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)]. In contrast, collagen induced massive production of PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3) in the presence of IAS. When testing the effect of each inhibitor individually we found the strongest inhibition of thrombin-induced PtdIns(3,4)P(2) production with the ADP scavenger system CP/CPK. Furthermore, we found a strong synergistic effect between exogenously added ADP and thrombin on production of PtdIns(3,4)P(2). In contrast to the results from 3-phosphorylated phosphoinositides, CP/CPK had little effect on thrombin-induced protein tyrosine phosphorylation. Our results show the importance of autocrine stimulation in thrombin-induced accumulation of 3-phosphorylated phosphoinositides and raise the question as to whether thrombin by itself is capable of inducing PI 3-K activation. In marked contrast to thrombin, collagen per se appears to be able to trigger increased production of PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3).

Direct demonstration of radiolabeled von Willebrand factor binding to platelet glycoprotein Ib and IIb-IIIa in the presence of shear stress.

In this study it is demonstrated for the first time that shear stress induces the binding of exogenous von Willebrand factor (vWF) multimers to platelets. The vWF preparations used were: 125I-vWF purified from human cryoprecipitate (and including all vWF multimers present in normal plasma); and 35S-cysteine-vWF secreted by human umbilical vein endothelial cells (HUVECs) (and containing unusually large vWF forms, as well as all plasma-type vWF multimers). Direct shear-induced binding to washed platelets (300-360 x 10(3)/microliters) of radiolabeled vWF was maximum at 60-120 dynes/cm2 evaluated at 30 sec and was in extent about one-quarter of the binding stimulated by ristocetin after 3 min of incubation. The shear-induced binding of only a small percentage of added radiolabeled vWF was sufficient to initiate aggregation. Radiolabeled vWF attached to both glycoprotein (GP) Ib and GPIIb-IIIa receptors in the shear field, with complete inhibition of binding occurring with simultaneous blockade of both receptors. Binding was potentiated by ADP released from sheared platelets.

Physical and chemical effects of red cells in the shear-induced aggregation of human platelets.

Both chemical and physical effects of red cells have been implicated in the spontaneous aggregation of platelets in sheared whole blood (WB). To determine whether the chemical effect is due to ADP leaking from the red cells, a previously described technique for measuring the concentration and size of single platelets and aggregates was used to study the shear-induced aggregation of platelets in WB flowing through 1.19-mm-diameter polyethylene tubing in the presence and absence of the ADP scavenger enzyme system phosphocreatine-creatine phosphokinase (CP-CPK). Significant spontaneous aggregation was observed at mean tube shear rates, (G) = 41.9 and 335 s-1 (42% and 13% decrease in single platelets after a mean transit time (t) = 43 s, compared to 89 and 95% decrease with 0.2 microM ADP). The addition of CP-CPK, either at the time of, or 30 min before each run, completely abolished aggregation. In the presence of 0.2 microM ADP, CP-CPK caused a reversal of aggregation at (t) = 17 s after 30% of single cells had aggregated. To determine whether red cells exert a physical effect by increasing the time of interaction of two colliding platelets (thereby increasing the proportion of collisions resulting in the formation of aggregates), an optically transparent suspension of 40% reconstituted red cell ghosts in serum containing 2.5-micron-diameter latex spheres (3 x 10(5)/microliters) flowing through 100-microns-diameter tubes was used as a model of platelets in blood, and the results were compared with those obtained in a control suspension of latex spheres in serum alone. Two-body collisions between microspheres in the interior of the flowing ghost cell or serum suspensions at shear rates from 5 to 90 s-1 were recorded on cine film. The films were subsequently analyzed, and the measured doublet lifetime, tau meas, was compared with that predicted by theory in the absence of interactions with other particles, tau theor. The mean (tau meas/tau theor) for doublets in ghost cell suspensions was 1.614 +/- 1.795 (SD; n = 320), compared to a value of 1.001 +/- 0.312 (n = 90) for doublets in serum. Whereas 11% of doublets in ghost cell suspensions had lifetimes from 2.5 to 5 times greater than predicted, in serum, no doublets had lifetimes greater than 1.91 times that predicted. There was no statistically significant correlation between tau meas/tau theor and shear rate, but the values of tau meas/tau theor for low-angle collisions in ghost cell suspensions were significantly greater than for high-angle collisions.

Necessity of newly synthesized ATP by creatine kinase for contraction of permeabilized longitudinal muscle preparations of rat proximal colon.

Necessity of newly synthesized ATP by creatine kinase for synthesis of ATP as an energy source for smooth muscle contraction was studied in permeabilized longitudinal muscle preparations of rat proximal colon. In alpha-toxin-permeabilized preparations, Ca++ induced "phasic type" contraction in a normal bath solution containing 4 mM ATP and 5 mM phosphocreatine. Omission of phosphocreatine from the solution resulted in significant decrease in phasic contraction, and omission of ATP resulted in loss of the response to Ca++. When ADP, but not adenosine-5-O-(2-thiodiphosphate), with phosphocreatine was added as a substitute for ATP, Ca++ induced the same type of contraction as with ATP. The maximum tensions of the phasic and tonic phases of the contraction with ADP were approximately 60% of, and almost the same, respectively as those with ATP. A selective inhibitor of creatine kinase, 2,4-dinitrofluorobenzene, inhibited the phasic contraction induced by Ca++. After irreversible inhibition of endogenous creatine kinase by DNFB in beta-escin-permeabilized preparations, treatment of the preparations with exogenous creatine kinase restored Ca(++)-induced contraction. These findings suggest that ATP synthesized from ADP and phosphocreatine by creatine kinase was necessary for phasic contraction of permeabilized smooth muscle and that exogenous ATP was mainly used after its hydrolysis to ADP.

Extracellular ATP inhibits agonist-induced mobilization of internal calcium in human platelets.

Our previous studies have demonstrated that platelets possess ATP purinergic receptors in addition to the ADP, P2T, receptor. Occupancy of the P2 receptor by ATP inhibited agonist-induced platelet aggregation. This study demonstrated that the mechanism of inhibition may involve ATP inhibition of agonist-induced mobilization of internal calcium. Within the cardiovascular system, the ATP inhibition of calcium mobilization is unique to platelets. All other cell types in the cardiovascular system, where calcium mobilization is affected by extracellular ATP, responded with an increased mobilization as opposed to inhibition. The platelet inhibitory response to ATP was enhanced by the addition of an ATP generating system, creatine phosphate/phosphocreatine kinase. ATP and ATP analogues were found to inhibit calcium mobilization with a rank order of alpha beta-methylene ATP, beta gamma-methylene ATP approximately ATP > benzoyl ATP > 2 methylthio ATP which is a characteristic of P2x-like receptors. The inhibitory effect of ATP could be abrogated by prolonged treatment of platelets with the P2x desensitizing agent, alpha beta-methylene ATP. Also, UTP and CTP were approximately as effective inhibitors as ATP while GTP was not. ATP competition with ADP for the P2T receptor was excluded in studies with platelets derived from an aspirin-treated individual which were essentially insensitive to ADP. The agonist-induced calcium mobilization and inhibition by ATP occurred with the thromboxane A2 mimetic, U46619, collagen and thrombin; however, the kinetics of mobilization varied somewhat with the different agonists. The responses to extracellular ATP were independent of extracellular Ca2+, where 1 mM calcium or 0.3 mM EGTA was added to the reaction mixture. The inhibition of calcium mobilization coupled to inhibition of platelet aggregation by extracellular ATP may serve an important physiologic role. ATP, released from activated platelets at localized sites of vascular injury, may help to limit the size of the platelet plug-clot that, if left unregulated, could occlude the injured blood vessel.

Role of cyclic nucleotides in rapid platelet adhesion to collagen.

Adhesion of human platelets to type I collagen under arterial flow conditions is extremely fast, being mediated primarily by the alpha 2 beta 1 integrin (glycoprotein Ia/IIa). We have investigated the involvement of cyclic nucleotides in platelet adhesion to soluble native collagen immobilized on Sepharose beads using a new microadhesion assay under arterial flow conditions. To prevent platelet stimulation by thromboxanes and adenosine diphosphate (ADP), experiments were performed with aspirin-treated platelets in the presence of ADP-removing enzyme systems such as creatine phosphate/creatine phosphokinase or apyrase. Rapid reciprocal changes in platelet adenosine 3'5'-cyclic monophosphate (cAMP) and guanosine 3'5'-cyclic monophosphate (cGMP) occurred during adhesion. cAMP levels in adherent platelets were 2.4-fold lower than in effluent platelets or in static controls, whereas cGMP levels were increased 2.4-fold. These results suggest that contact between platelets and collagen stimulates guanylate cyclase and inhibits adenylate cyclase. This occurs in the absence of the platelet release reaction. We also studied short-term effects of agents that regulate cyclic nucleotide synthesis, prostaglandin E1 (PGE1) and sodium nitroprusside (SNP). After only 3.8 seconds at 10 to 30 dyne/cm2, PGE1 (10 mumol/L) increased cAMP 16.4-fold, whereas SNP (50 mumol/L) increased cGMP ninefold and caused a 3.2-fold increase in cAMP. Both PGE1 and SNP rapidly (< 5 seconds) inhibited platelet adhesion in a dose-dependent manner that was correlated with the increase in cyclic nucleotides. Our data suggest that cAMP and cGMP play a regulatory role in the initial phases of platelet adhesion to collagen mediated by the alpha 2 beta 1 integrin receptor.