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Reducing myocardial damage resulting from ischaemia-reperfusion (I/R) is vital in ensuring patient recovery and survival. It relies upon the activation of the so-called Reperfusion Injury Salvage Kinase (RISK) pathway. Experimentally various treatments, both mechanical and chemical, have been shown to protect the myocardium against I/R injury. Chemical facilitators of myocardial preservation include endogenous factors such as insulin, erythropoietin and glucagon-like peptide 1. The adipocytokines, products of white adipose tissue, are important peptide hormones with respect to metabolic control and satiety, and were formerly considered in the context of obesity and metabolic disease. More recently, however, evidence has been presented indicating that the adipocytokines play significant roles in cardiac function and, as we have suggested, in myocardial protection. To date leptin, adiponectin, apelin and visfatin have all been shown to protect against I/R injury. Significantly, the protection afforded by these peptides involves the activation of kinases which are key elements of the mechanisms underlying tissue preservation, including the RISK pathway components PI3K-Akt and p44/42, and inhibition of the mitochondrial permeability transition pore (MPTP). In this article we examine the roles played by the adipocytokines in cardiovascular function and disease. In particular, we focus on the evidence that these peptides promote myocardial survival, much of it having been obtained in this laboratory. To conclude, we discuss some future directions in the field, including the prospects for some of the adipocytokines finding application as therapeutic agents in myocardial infarction.

Leptin-induced protection against myocardial ischemia-reperfusion (I/R) injury involves the activation of the reperfusion injury salvage kinase pathway, incorporating phosphatidylinositol 3-kinase-Akt/protein kinase B and p44/42 MAPK, and the inhibition of the mitochondrial permeability transition pore (MPTP). Recently published data indicate that the JAK/STAT signaling pathway, which mediates the metabolic actions of leptin, also plays a pivotal role in cardioprotection. Consequently, in the present study we considered the possibility that JAK/STAT signaling linked to the MPTP may be involved in modulating the cardioprotective actions of leptin. Employing rat in vitro models (Langendorff-perfused hearts and cardiomyocytes) of I/R injury, we investigated the actions of leptin (10 nM), administered at reperfusion, in the presence or absence of the JAK2 inhibitor, AG-490 (5 μM). Leptin reduced infarct size significantly (control, 60.05 ± 7.41% vs. leptin treated, 29.9 ± 3.24%, P < 0.05), protection being abolished by AG-490. Time course studies revealed that leptin caused a 171% ( P < 0.001) increase in STAT3/tyrosine-705 phosphorylation at 2.5 min reperfusion; however, increases were not seen at 5, 10, 15, or 30 min reperfusion. Contrasting with STAT3, Akt/serine-473 phosphorylation was not significantly increased until 15 min into the reperfusion phase (140%, P < 0.05). AG-490 blocked the leptin-induced rise in STAT3 phosphorylation seen at 2.5 min reperfusion but did not influence Akt/serine-473 phosphorylation at 15 min. Leptin reduced the MPTP opening ( P < 0.001), which was blocked by AG-490. This is the first study to yield evidence that JAK/STAT signaling linked to the MPTP plays a role in leptin-induced cardioprotection. Under the experimental conditions employed, STAT3 phosphorylation appears to have occurred earlier during reperfusion than that of Akt. Further research into the interactions between these two signaling pathways in the setting of I/R injury is, however, required.

CB1 antagonism is associated with reduced doxorubicin-induced cardiotoxicity and decreased cerebrocortical infarction. Rimonabant, a selective CB1 receptor antagonist, was, before it was withdrawn, proposed as a treatment for obesity and reported to reduce cardiovascular risk by improving glucose and lipid profiles and raising adiponectin levels. The cardioprotective actions of rimonabant in 6-week-old C57BL/6J mice fed either high-fat (HFD) or standard diets (STD) for 8 weeks were investigated. At 14 weeks, mice received rimonabant (10 mg/kg/day, i.p.) or vehicle for 1 week and were then subjected to an in vivo acute myocardial infarction. The influence of rimonabant on infarct size (IS) in CB1 knockout (CB1-/-) and wild-type (CB1+/+) mice was also examined. C57BL/6J mice that had been maintained on STD or HFD exhibited 4.3 and 21.4% reductions in body weight following 7 days rimonabant treatment. Rimonabant reduced IS in both STD (29.6 +/- 3.5% vs. 49.8 +/- 6.9% in control, P0.05) and HFD (26.9 +/- 1.5% vs. 48.7 +/- 7% in control, P0.05) mice. In CB1-/- mice rimonabant failed to reduce body weight or IS (51.0 +/- 5.3% vs. 49.7 +/- 4.7% in control, P0.05), although significant reductions were seen in CB1+/+ mice (IS, 48.9 +/- 4.6% control vs. 30.5 +/- 3.1% rimonabant, P0.05). To exclude the possibility that weight loss alone induced cardioprotection, HFD mice were switched to STD for 7 days (HFD-STD), resulting in an 11.3 +/- 1.0% decrease in body weight compared to control (+2.1 +/- 1.1% in HFD). This, however, was not associated with IS reduction (39.1 +/- 3.9% HFD-STD vs. 40.0 +/- 5.3% HFD, P0.05). Serum and cardiac adiponectin levels were unaltered by rimonabant treatment. HL-1 cell death was not prevented by 1 or 7 days treatment with rimonabant. We conclude that rimonabant-induced infarct limitation may involve the CB1 receptor, although not necessarily cardiac CB1 receptors, and is unrelated to weight loss or altered adiponectin synthesis.

Visfatin is an adipocytokine capable of mimicking the glucose-lowering effects of insulin and activating the pro-survival kinases phosphatidylinositol-3-OH kinase (PI3K)-protein kinase B (Akt) and mitogen-activated protein kinase kinase 1 and 2 (MEK1/2)-extracellular signal-regulated kinase 1 and 2 (Erk 1/2). Experimental studies have demonstrated that the activation of these kinases confers cardioprotection through the inhibition of the mitochondrial permeability transition pore (mPTP). Whether visfatin is capable of exerting direct cardioprotective effects through these mechanisms is unknown and is the subject of the current study. Anaesthetized C57BL/6 male mice were subjected to in situ 30 min. of regional myocardial ischaemia and 120 min. of reperfusion. The administration of an intravenous bolus of visfatin (5 × 10−6μmol) at the time of myocardial reperfusion reduced the myocardial infarct size from 46.1 ± 4.1% in control hearts to 27.3 ± 4.0% (n≥ 6/group, P < 0.05), an effect that was blocked by the PI3K inhibitor, wortmannin, and the MEK1/2 inhibitor, U0126 (48.8 ± 5.5% and 45.9 ± 8.4%, respectively, versus 27.3 ± 4.0% with visfatin; n≥ 6/group, P < 0.05). In murine ventricular cardiomyocytes subjected to 30 min. of hypoxia followed by 30 min. of reoxygenation, visfatin (100 ng/ml), administered at the time of reoxygenation, reduced the cell death from 65.2 ± 4.6% in control to 49.2 ± 3.7%(n > 200 cells/group, P < 0.05), an effect that was abrogated by wortmannin and U0126 (68.1 ± 5.2% and 59.7 ± 6.2%, respectively; n > 200 cells/group, P > 0.05). Finally, the treatment of murine ventricular cardiomyocytes with visfatin (100 ng/ml) delayed the opening of the mPTP induced by oxidative stress from 81.2 ± 4 sec. in control to 120 ± 7 sec. (n > 20 cells/group, P < 0.05) in a PI3K- and MEK1/2-dependent manner. We report that the adipocytokine, visfatin, is capable of reducing myocardial injury when administered at the time of myocardial reperfusion in both the in situ murine heart and the isolated murine cardiomyocytes. The mechanism appears to involve the PI3K and MEK1/2 pathways and the mPTP.

Activation of the Reperfusion Injury Salvage Kinase (RISK) pathway, which incorporates phosphatidylinositol-3-OH kinase (PI3K)-Akt/protein kinase B (PKB) and p44/42 mitogen-activated protein kinase (MAPK), underlies protection against ischemia-reperfusion (I/R) injury. The temporal nature of the activation of these RISK pathway components during reperfusion is, however, uncertain. We examined Akt and p44/42 phosphorylation in hearts subjected to ischemia and varying periods of reperfusion in the absence or presence of the putative cardioprotectant, apelin-13. Akt activity was also measured.Langendorff perfused C57Bl/6J mouse hearts were subjected to 35 min global ischemia followed by 0, 2.5, 5 or 10 min reperfusion with or without 1 microM apelin-13. Basal and apelin-induced phosphorylation of Akt (at both the threonine 308 and serine 473 phosphorylation sites) and p44/42 during the reperfusion phase was determined by Western blotting and Akt activity measured using an Enzyme-Linked ImmunoSorbent Assay (ELISA).Basal phosphorylation of both Akt and p44/42 increased progressively with time of reperfusion. Apelin enhanced Akt and p44/42 phosphorylation at all reperfusion time points. Akt activity did not change under basal conditions but was increased by apelin at 5 min (NS) and 10 min (p0.05) reperfusion.We conclude that under basal conditions Akt and p44/42 phosphorylation increases with time of reperfusion but that this is not accompanied by increased kinase (Akt) activity. On application of a cardioprotectant, however, kinase phosphorylation and activity are enhanced suggesting that it is the combination of these two mechanisms that may underly the tissue preserving actions of such agents.

Necrostatin-1 (Nec-1), a small tryptophan-based molecule, was recently reported to protect the cerebral cortex against ischemia-reperfusion (I/R) injury. We investigated the actions of Nec-1 and its so-called inactive analog, Nec-1i, in the setting of myocardial I/R injury.The actions of Nec-1 and Nec-1i were examined in cultured C2C12 and H9c2 myocytes, cardiomyocytes isolated from male Sprague-Dawley rats, Langendorff isolated perfused C57Bl/6J mouse hearts and an in vivo open-chest C57Bl/6J mouse heart model.Nec-1 at 30 microM and 100 microM (but not 100 microM Nec-1i) reduced peroxide-induced cell death in C2C12 cells from 51.2 +/- 1.1% (control) to 26.3 +/- 2.9% (p0.01 vs control) and 17.8 +/- 0.9% (p0.001), respectively. With H9c2 cells cell death was also reduced from 73.0 +/- 0.4% (control) to 56.7 +/- 0% (30 microM Nec-1, p0.05) and 45.4 +/- 3.3% (100 microM Nec-1, p0.01). In the isolated perfused heart Nec-1 (30 microM) reduced infarct size (calculated as a percentage of the risk area) from 48.0 +/- 2.0% (control) to 32.1 +/- 5.4% (p0.05). Nec-1i (30 microM) also reduced infarct size (32.9 +/- 5.1%, p0.05). In anesthetized C57Bl/6J mice Nec-1 (1.65 mg/kg), given intraperitoneally to coincide with reperfusion following left anterior descending artery ligation (30 min), also reduced infarct size from 45.3 +/- 5.1% (control) to 26.6 +/- 4.0% (p0.05), whilst Nec-1i (1.74 mg/kg) was ineffective (37.8 +/- 6.0%). Stimulus-induced opening of the mitochondrial permeability transition pore (MPTP) in rat cardiomyocytes, as reflected by the time until mitochondrial depolarisation, was unaffected by Nec-1 or Nec-1i at 30 muM but increased at 100 muM i.e. 91% (p0.05 vs control) and 152% (p0.001) for Nec-1 and Nec-1i, respectively.This is the first study to demonstrate that necrostatins inhibit myocardial cell death and reduce infarct size, possibly via a mechanism independent of the MPTP.

Background and purpose: Protection against ischaemia-reperfusion (I/R) injury involves PI3K-Akt and p44/42 MAPK activation. Leptin which regulates appetite and energy balance also promotes myocyte proliferation via PI3K-Akt and p44/42 MAPK activation. We, therefore, hypothesized that leptin may also exhibit cardioprotective activity. Experimental approach: The influence of leptin on I/R injury was examined in perfused hearts from C57Bl/6 J mice that underwent 35 min global ischaemia and 35 min reperfusion, infarct size being assessed by triphenyltetrazolium chloride staining. The concomitant activation of cell-signalling pathways was investigated by Western blotting. The effect of leptin on mitochondrial permeability transition pore (MPTP) opening was studied in rat cardiomyocytes. Key results: Leptin (10 nM) administered during reperfusion reduced infarct size significantly. Protection was blocked by either LY294002 or UO126, inhibitors of Akt and p44/42 MAPK, respectively. Western blotting confirmed that leptin stimulated p44/42 MAPK phosphorylation significantly. Akt phosphorylation was also enhanced but did not achieve statistical significance. Additionally, leptin treatment was associated with a significant increase in p38 phosphorylation. By contrast, leptin caused downregulation of phosphorylated and non-phosphorylated STAT3, and of total AMP-activated kinase. Cardiomyocytes responded to leptin with delayed opening of the MPTP and delayed time until contracture. Conclusions and implications: Our data indicate for the first time that the adipocytokine, leptin, has direct cardioprotective properties which may involve the PI3-Akt and p44/42 MAPK pathways. British Journal of Pharmacology (2006) 149, 5–13. doi:10.1038/sj.bjp.0706834

Soluble beta-amyloid (A beta) 40 peptide (1 microM) has been reported to enhance phenylephrine and endothelin-1 induced contraction of rat aortic rings. We conducted similar experiments with aortic rings from Sprague-Dawley (SD), Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats but employing noradrenaline (NA) as the vasoconstrictor. Unlike previous studies we found that, rather than enhancing agonist-induced contraction, 1 microM A beta 40 attenuated the vasoconstrictive responses to NA. With aortic rings from SD rats the attenuation of contractile responses was coupled with a 99% increase in NA EC(50) values. EC(50) values obtained for aortic rings from WKY and SHR, however, exhibited no changes. Contrasting with the effects observed with 1 microM A beta 40, treatment of SD aortic rings with 5 microM A beta 40 resulted in potentiation of NA-induced constriction and a 46% decrease in EC(50) values. We hypothesise that at low concentrations A beta 40 may cause attenuation of NA-induced constriction by dint of enhanced endothelial vasodilator (nitric oxide, prostacyclin) synthesis. By contrast, at higher concentrations A beta 40 may potentiate vasoconstriction via the generation of toxic A beta oligomers which act on the endothelium to reduce vasodilator output.

Alzheimer's disease (AD) is characterised by the accumulation of insoluble beta-amyloid (A beta) fibrils in the brain. Factors that promote A beta fibrillogenesis may influence the pathogenesis of AD and represent targets for therapeutic intervention. Some A beta deposited in AD may originate in the circulation and plasma factors could promote A beta deposition, particularly in the cerebrovasculature. We investigated the effects of plasma low-density lipoprotein (LDL), in both its native and oxidised forms, on A beta(1-40) fibrillogenesis and vasoactivity. LDL enhanced A beta fibrillogenesis in a process dependent on LDL concentration and the oxidative state of the lipoprotein, as indicated by measurements of thiobarbituric acid reactive substances (TBARS) and conjugated dienes. LDL's actions were inhibited by the iA beta 5 peptide, suggesting that LDL-induced A beta polymerisation involved beta-pleated sheet formation. Potentiated A beta polymerisation was reflected by enhanced A beta-mediated vascular responses. Human endothelial cells exposed to fibrillar A beta generated with LDL, especially oxidised LDL, exhibited decreased 20S proteasome activity. Rat aortic ring constriction induced by noradrenaline was enhanced by A beta fibrils generated with LDL, with oxidised LDL producing the more marked effects. Should plasma lipoproteins prove to play a role in cerebral A beta deposition their modification with statins or antioxidants may offer therapeutic benefit.

The toxicity of the beta-amyloid (Abeta) peptide of Alzheimer's disease may relate to its polymerisation state (i.e. fibril content). We have shown previously that plasma lipoproteins, particularly when oxidised, greatly enhance Abeta polymerisation. In the present study the nature of the interactions between both native and oxidised lipoproteins and Abeta1-40 was investigated employing various chemical treatments. The addition of ascorbic acid or the vitamin E analogue, trolox, to lipoprotein/Abeta coincubations failed to inhibit Abeta fibrillogenesis, as did the treatment of lipoproteins with the aldehyde reductant, sodium borohydride. The putative lipid peroxide-derived aldehyde scavenger, aminoguanidine, however, inhibited Abeta-oxidised lipoprotein-potentiated polymerisation, but in a manner consistent with an antioxidant action for the drug. Lipoprotein treatment with the reactive aldehyde 4-hydroxy-2-trans-nonenal enhanced Abeta polymerisation in a concentration-dependent fashion. Incubation of Abeta with lipoprotein fractions from which the apoprotein components had been removed resulted in extents of polymerisation comparable to those observed with Abeta alone. These data indicate that the apoprotein components of plasma lipoproteins play a key role in promoting Abeta polymerisation, possibly via interactions with aldehydes.

Background: Nitric oxide (NO), synthesised from the inducible isoform of nitric oxide synthase (iNOS), is implicated in mediating second window of protection (SWOP)/delayed ischemic preconditioning. However the role of NO and iNOS in delayed pharmacological protection remains unclear and is the subject of this investigation. Methods: To test the hypothesis that iNOS is necessary for delayed pharmacological preconditioning, the adenosine A1 receptor agonist, 2-chloro N6 cyclopentyl adenosine (CCPA) (25 μg/kg i.v.) or saline was administered to wild type (WT) or iNOS gene knockout mice (KO). Twenty-four hours later, the hearts were isolated, Langendorff perfused and subjected to 35 min ischemia/30 min reperfusion prior to infarct size determination. Results: WT and KO control hearts had identical infarct sizes of 37±3% and 37±2%, respectively. CCPA significantly reduced infarct size in WT hearts to 22±2% and also, unexpectedly, in KO hearts (27±2%). This protection was abrogated with the non-specific NOS inhibitor, Nω nitro l-arginine methyl ester (l-NAME, 100 μM), and could be mimicked in naive hearts with the NO donor, donor S -nitroso N -acetyl dl penicillamine (SNAP, 1 μM). Delayed protection appeared to be mediated by NO synthesis in both WT and KO hearts. Additional studies using Western blot analysis demonstrated endothelial NOS (eNOS) upregulation and increased NO x release in both WT and KO hearts. Conclusions: This is the first study to demonstrate a role for eNOS in delayed A1 receptor triggered (pharmacological) preconditioning, potentially representing a new pharmacological target for protecting the ischemic heart.

Collagen (5-160 microg/ml) induced release of free and sulphate conjugated noradrenaline (NA), adrenaline (Ad) and dopamine (DA) by platelets in platelet-rich plasma (PRP) was investigated in human subjects. Stimulated efflux of free NA, Ad and DA to the plasma (platelet-poor plasma; PPP) increased in a concentration-dependent manner, NA release being reflected by reduced platelet free NA contents. Collagen EC(50) values for free NA, Ad and DA release were similar, i.e. 3.8, 3.3 and 4.5 microg/ml collagen, respectively. Subtracting resting PPP free catecholamine (CA) concentrations from those obtained with 160 microg/ml collagen revealed that 52%, 31% and 33% of PPP free NA, Ad and DA, respectively, resulted from collagen stimulation. Under resting conditions sulphate conjugated NA, Ad and DA accounted for 74%, 78% and 99% of total (sum of free and sulphate conjugated) PPP concentrations. Collagen elicited concentration-dependent release of sulphate conjugated DA and, to a lesser extent, NA, but not Ad. Sulphate conjugated NA release was mirrored by decreases in platelet sulphate conjugated NA contents. Resting platelet sulphate conjugated NA and Ad represented 42% and 50% of total (free plus sulphate conjugated) NA and Ad concentrations. EC(50) values for sulphate conjugated DA and NA release were, respectively, 14.5 and 6.25 microg/ml collagen, which exceeded the values for free DA and NA release by 222% and 64%. With 160 microg/ml collagen 44% and 15% of PPP sulphate conjugated DA and NA, respectively, had resulted from platelet activation, and of the total (sum of free and sulphate conjugated) DA and NA released, 99.5% and 39.7% were sulphate conjugated. Total (sum of PPP and platelet) free and sulphate conjugated NA and Ad concentrations were unaltered by collagen stimulation indicating that platelet activation does not cause mobilisation (i.e. hydrolysis) of platelet sulphate conjugated CA. Thus, platelet activation causes the liberation of sulphate conjugated CA (particularly DA) as well as free CA. The differences with regard to the collagen EC(50) values for free and sulphate conjugated CA release indicate that these species arise from different subcellular compartments.

Serotonin (5-HT) and the catecholamines (CA), noradrenaline (NA) and adrenaline (Ad), are platelet dense-granule constituents which influence platelet activity and vessel tone. Platelets accumulate 5-HT via an active process whilst CA uptake occurs mainly by passive diffusion. The platelet contents and collagen-stimulated efflux of 5-HT, NA and Ad were examined in normal individuals to establish whether relationships exist between these monoamines. Regression analysis revealed that platelet 5-HT was not related to platelet NA or Ad levels. Platelet NA, however, correlated positively with Ad (r = 0.61, P0.01). Collagen-induced release of all three monoamines occurred in a dose-dependent manner. The collagen EC50 values for 5-HT and CA release, however, differed and were greater for 5-HT release: 9.6 +/- 0.8 vs. 3.8 +/- 0.2 microgram/ml collagen, 5-HT vs. NA, P0.001; 9.6 +/- 0.8 vs. 3.9 +/- 0.5 microgram/ml, 5-HT vs Ad, P0.001. These data may reflect differences regarding the triggering mechanisms for 5-HT and CA release and provide evidence for separate compartments of intra-platelet 5-HT and CA and possibly distinct populations of 5-HT and CA containing dense granules and/or platelets.

Platelet sensitivity to ADP and adrenaline was determined after storage of platelet-rich plasma (PRP) under various conditions to establish those yielding optimal platelet stability. The effects of the exclusion of air from the storage syringes, temperature, PRP dilution and duration of storage were tested. Storage at room temperature (22° C) in the absence of air stabilised PRP pH over 24 h and stabilised platelet sensitivity to ADP up to 4 h. Storage at 4°C and 13 C caused platelet activation and eventually spontaneous aggregation, as evidenced by significant reductions in platelet counts. Samples stored at 37° C were less responsive to ADP and adrenaline than samples maintained at 22 C. Platelet count adjustment to 200 × 10(9)/L reduced platelet sensitivity as reflected by increased agonist EC(50) values and threshold concentrations. Positive correlations between agonist EC(50) values (and between threshold concentrations) for diluted and undiluted samples were obtained, indicating that platelet count adjustment did not affect the ranking order of platelet sensitivity within the subject group. No correlations between platelet count and indices of platelet sensitivity were seen suggesting that differences in platelet aggregation arise from intrinsic differences in platelet sensitivity rather than differences in platelet count. With time of storage the responses to ADP (EC(50) and threshold concentration) and adrenaline (EC(50)) declined to a greater extent for undiluted PRP than for diluted PRP. No changes in the platelet-poor plasma concentrations of the dense granular component, serotonin, occurred in diluted or undiluted samples over 24 h. We conclude that in order to ensure optimal stability of platelets, PRP should be stored at room temperature (22°C) in the absence of air and tested within 4 h of preparation. A decision on platelet count adjustment is also required dependent upon the experimental objectives.

Cell death is an integral part of the life of an organism being necessary for the maintenance of organs and tissues. If, however, cell death is allowed to proceed unrestricted, tissue damage and degenerative disease may ensue. Until recently, three morphologically distinct types of cell death were recognized, apoptosis (type I), autophagy (type II) and necrosis (type III). Apoptosis is a highly regulated, genetically determined mechanism designed to dismantle cells systematically (e.g. cells that are no longer functionally viable), via protease (caspase) action, and maintain homeostasis. Autophagy is responsible for the degradation of cytoplasmic material, e.g. proteins and organelles, through autophagosome formation and subsequent proteolytic degradation by lysosomes, and is normally considered in the context of survival although it is sometimes associated with cell death. Necrosis was formerly considered to be an accidental, unregulated form of cell death resulting from excessive stress, although it has been suggested that this is an over-simplistic view as necrosis may under certain circumstances involve the mobilization of specific transduction mechanisms. Indeed, recently, an alternative death pathway, termed necroptosis, was delineated and proposed as a form of ‘programmed necrosis’. Identified with the aid of specific inhibitors called necrostatins, necroptosis shares characteristics with both necrosis and apoptosis. Necroptosis involves Fas/tumour necrosis factor-α death domain receptor activation and inhibition of receptor-interacting protein I kinase, and it has been suggested that it may contribute to the development of neurological and myocardial diseases. Significantly, necrostatin-like drugs have been mooted as possible future therapeutic agents for the treatment of degenerative conditions.

The aim of the study was to determine whether aspirin influences sympathoadrenal output in normal human subjects. Plasma and platelet adrenaline and noradrenaline levels were measured before and after chronic administration of oral aspirin (300 mg per day for 7 days). Catecholamine concentrations measured immediately following aspirin did not differ from control (pre-treatment) values. Platelet noradrenaline and plasma adrenaline levels were, however, significantly increased 2 weeks after cessation of treatment. Platelet TxB2 generation was significantly reduced following aspirin treatment indicating that platelet cyclooxygenase had been inhibited. Catecholamine concentrations did not correlate with TxB2 generation. In vitro platelet aggregation induced by ADP, adrenaline and collagen was reduced after aspirin providing additional confirmation of cyclooxygenase inhibition. However, the in vivo markers of platelet function, beta-TG and PF4 were unaffected. These data do not provide convincing evidence for an action of aspirin on sympathoadrenal outflow, either directly or via a prostaglandin (thromboxane) mediated effect, although this does not exclude a later, delayed effect. There was no evidence for interactions between thromboxane, catecholamine levels in plasma and platelets, and platelet function.

Experimental studies have linked the adipocytokines with acute cardioprotection. Whether the adipocytokine, resistin, confers protection is, however, debatable. In the current study, the actions of resistin, administered at reperfusion, were investigated in in vivo and in vitro rodent and in vitro human models of myocardial ischemia-reperfusion (I/R) injury. Resistin did not reduce infarct size in Langendorff-perfused rat hearts or murine hearts perfused in vivo. Resistin also did not protect human atrial muscle subjected to hypoxia-reoxygenation. Although cyclosporin A delayed mitochondrial permeability transition pore (MPTP) opening in murine cardiomyocytes, resistin was ineffective. Western blot analysis revealed that resistin treatment was associated with enhanced phosphorylation of Akt, at both the serine-473 (+ 51.9%, P = .01) and threonine-308 (+107%, P < .01) phosphorylation sites, although not to the extent seen with ischemic preconditioning (+132.5%, P = .002 and +389.1%, P < .01, respectively). We conclude that resistin administered at reperfusion at concentrations/doses equivalent to normal (upper end) and pathological serum levels does not protect against I/R injury or inhibit MPTP opening.

Insulin resistance and hyperinsulinemia is now recognized in non-insulin-dependent diabetes, essential hypertension, obesity, atherosclerotic heart disease, dyslipidemia, heart failure, and in heavy smokers. Several mechanisms have been proposed to explain hyperinsulinemia, insulin resistance and its relationship to hypertension; reduced sodium excretion, activation of the sympathetic nervous system, increased activity of the sodium/hydrogen pump, and stimulation of cellular growth. Some of the nonpharmacological methods to control hyperinsulinemia are of benefit in the management of hypertension, most notably weight loss, exercise program, and reduced salt intake. High-fiber and reduced-protein diets also reduce hyperinsulinemia. Thiazide diuretics can result in insulin resistance, and insulin secretion may be inhibited, possibly associated with concomitant hypokalemia. beta-Blockers result in some reduction of glucose tolerance and mask some of the features of hypoglycemia. Angiotensin-converting enzyme (ACE) inhibitors and alpha-receptor blockers do not effect insulin resistance; probably the same is true for calcium antagonists. Although the effect on risk factors should not be discounted, it is the effect of treatment on hard end points, cerebrovascular accidents, myocardial infarction, or death that is most important. Evidence in hypertension is at present restricted to diuretics and beta-blocking drugs.

beta-amyloid (Abeta) arises from an amyloid precursor protein (APP) via beta- and gamma-secretase proteolysis. Platelets are the primary depot of APP in the circulation and may be a source of cerebral Abeta. We measured the platelet activities of alpha- and beta-secretases in normal individuals. Platelet alpha- and beta-secretase activities were linear with respect to incubation time and quantity of platelet extract included in the assays. beta-secretase activity, calculated relative to platelet count (PC) and platelet protein (PP), varied between individuals 3.5-fold and 4.6-fold, respectively. Meanwhile, alpha-secretase varied 3.75-fold and 5.8-fold. beta-secretase/alpha-secretase ratios varied 4.1-fold (PC) or 10.5-fold (PP). beta-secretase correlated with age (r = 0.779, p < 0.02, PP) and mean platelet volume (MPV) (r = 0.702, p < 0.05, PC). alpha-secretase correlated negatively with high-density lipoprotein (HDL) (-0.76, p < 0.01, PP). beta-secretase/alpha-secretase ratios correlated with MPV (r = 0.67, p < 0.05, PC) and HDL (r = 0.77, p < 0.02, PP). These data could indicate that platelet secretase activity is related to aging, platelet size and plasma lipoprotein levels. Clearly, however, this is a preliminary study involving a limited number of individuals and further, more detailed, investigations are required to establish that such relationships are valid.

Necrostatin (Nec-1) protects against ischemia-reperfusion (IR) injury in both brain and heart. We have previously reported in this journal that necrostatin can delay opening of the mitochondrial permeability transition pore (MPTP) in isolated cardiomyocytes. The aim of the present study was to investigate in more detail the role played by the MPTP in necrostatin-mediated cardioprotection employing mice lacking a key component of the MPTP, namely cyclophilin-D. Anaesthetized wild type (WT) and cyclophilin-D knockout (Cyp-D-/-) mice underwent an open-chest procedure involving 30 minutes of myocardial ischemia and 2 hours of reperfusion, with subsequent infarct size assessed by triphenyltetrazolium staining. Nec-1, given at reperfusion, significantly limited infarct size in WT mice (17.7±3% vs. 54.3±3%, P0.05). In conclusion, the data obtained in Cyp-D−/− mice provide further evidence that Nec-1 protects against myocardial IR injury by modulating MPTP opening at reperfusion.

Protection against myocardial ischemia-reperfusion (I/R) injury involves activation of phosphatidylinositol-3-OH kinase (PI3K)- Akt/protein kinase B and p44/42 mitogen-activated protein kinase (MAPK), components of the reperfusion injury salvage kinase (RISK) pathway. The adipocytokine, apelin, activates PI3K-Akt and p44/42 in various tissues and we, therefore, hypothesised that it might demonstrate cardioprotective activity. Employing both in vivo (open-chest) and in vitro (Langendorff and cardiomyocytes) rodent (mouse and rat) models ofmyocardial I/R injury we investigated if apelin administered at reperfusion at concentrations akin to pharmacological doses possesses cardioprotective activity. Apelin-13 and the physiologically less potent peptide, apelin-36, decreased infarct size in vitro by 39.6% (p

Cerebrovascular accumulation of A beta (beta-amyloid) occurs in aging and AD (Alzheimer's disease). Hypercholesterolaemia, which is associated with raised plasma LDL (low-density lipoprotein), may predispose to AD. Soluble A beta is found in the circulation and enhances vasoconstriction. Under conditions that may favour the formation of short A beta oligomers, as opposed to more severe polymerization leading to A beta fibrillogenesis, we investigated the influence of LDLs on the vasoactive actions of soluble A beta. Thus the actions of A beta 40 and A beta 42 in combination with native or,oxidized LDL on vasoconstriction to NA (noradrenaline) and vasodilatation to ACh (acetylcholine) were examined in rat aortic rings. LDL, particularly when oxidized, potentiated NA-induced constriction when combined with soluble A beta 40 and, especially, A beta 42. Soluble A beta 40 reduced relaxation induced by ACh, but A beta 42 was ineffective. Native and oxidized LDL also attenuated relaxation. Synergism occurred between oxidized LDL and A beta with respect to ACh-induced relaxation, but not between native LDL and A beta. We have shown for the first time that, under conditions that may result in A beta oligomer formation, LDL, particularly when oxidized, modulates the vascular actions of soluble A beta to extents greater than those reported previously for fibrillar A beta preparations. Mechanisms whereby a treatable condition, namely hypercholesterolaemia, might contribute to the development of the cerebrovascular component of AD are indicated.

The circulatory system is a potential source of the beta-amyloid protein (A beta) of ageing and Alzheimer's disease (AD), platelets accounting for the bulk of A beta immunoreactivity detectable in blood. Evidence for the release of A beta by platelets, however, has not been reported. Platelets from normal donors were therefore stimulated with collagen to establish if A beta immunoreactive material is released on activation. For comparison, the release of the platelet monoamines, serotonin (5-HT) adrenaline (Adr) and noradrenaline (NA) was also measured. Like the monoamines, collagen-induced A beta release was concentration-dependent, maximal stimulated release exceeding basal efflux by 184%. Collagen EC50 values for A beta release were similar to those for Adr and NA (3.6 +/- 0.6, 3.4 +/- 0.6 and 3.3 +/- 0.2 microg/ml collagen, respectively) but not 5-HT (9.8 +/- 1.9 microg/ml). These data provide the first evidence that platelets release A beta immunoreactive material on stimulation and may indicate that A beta, Adr and NA reside in the same subcellular compartment.

The cytotoxic beta-amyloid peptide (Abeta) of Alzheimer's disease (AD) occurs in both plasma and platelets and may modulate platelet function. Its biological activity may relate to its fibril content and factors that promote Abeta fibrillogenesis, e.g., plasma lipoproteins could, therefore, have implications for Abeta action. We undertook a study in which structure-activity relationships were considered with respect to the actions of Abeta(1-40) on platelet function. Thus, the influence of soluble Abeta and various fibrillar Abeta preparations (0.1-10 microM) on platelet aggregation and endogenous 5-hydroxytryptamine (5-HT) efflux was investigated. Soluble Abeta(1-40) only enhanced platelet aggregation (+30%, P

The circulation constitutes a potential source of the beta-amyloid (A beta) protein deposited cerebrally in Alzheimer's disease (AD). Cardiovascular risk factors, including hyperlipidaemia, may be involved in the pathogenesis of AD. Plasma A beta 40 was measured by radioimmunoassay in normal and hyperlipidaemic subjects with the aim of determining if plasma lipid content and/or age correlated with circulating A beta 40 concentration. Plasma A beta 40 levels in hyperlipidaemics were elevated by 20.3% compared to normal subjects. A beta 40 did not correlate with plasma lipids in normal subjects. Age, however, correlated positively with A beta 40 in these individuals and with total cholesterol, low-density lipoprotein (LDL) and triglycerides. No correlations were observed in hyperlipidaemic patients or when the data for the two groups were combined. These data are consistent with ageing, the primary risk factor for AD, but not hyperlipidaemia influencing circulating A beta 40 levels.

Measurements of nitrite (NO(2)(-)) and nitrate (NO(3)(-)) in biological fluids are proposed as indices of cellular nitric oxide (NO) production. Determination of NO(2)(-) and NO(3)(-) in standard solutions is not difficult, however, determinations which reflect accurately cellular NO synthesis represent a considerable analytical challenge. Problems are often encountered arising from background NO(2)(-)/NO(3)(-) contamination in experimental solutions and laboratory hardware, and with methods for sample extraction. We investigated potential procedures for the extraction and determination of NO(2)(-) and NO(3)(-) in biological samples. Consequently, a protocol was devised which yielded acceptable results regarding extraction efficiency, assay reproducibility, sample throughput and contaminant minimisation. It entailed rigorous washing of all equipment with water of low NO(2)(-) and NO(3)(-) content, sample deproteinisation by centrifugal ultrafiltration through a 3K filter and analysis by high-performance anion-exchange liquid chromatography with UV detection. Retention times for NO(2)(-) and NO(3)(-) in standards and plasma were 4.4 and 5.6 min, respectively. Assay linearity for standards ranged between 31 nM and 1 mM. The limit of detection for NO(2)(-) and NO(3)(-) in standards was 3 pmol. Recoveries of NO(2)(-) and NO(3)(-) from spiked plasma (1-100 microM KNO(2)/KNO(3)) and from extracted standards (1-250 microM) were approximately 100%. Intra-assay and inter-assay RSDs for NO(2)(-) and NO(3)(-) in spiked and unspiked plasma were 10.6% or less. Assays on washed platelet supernatants demonstrated collagen-induced platelet generation of NO products and analysis of murine and rat cardiac perfusates was achieved. Our procedure may be suitable for routine determination of NO(2)(-) and NO(3)(-) in various biological fluids, e.g., plasma.

Tissue accumulation of the cytotoxic beta-amyloid peptide (Abeta) occurs in Alzheimer's disease (AD), one possible source being the platelet. AD and cardiovascular disease may share some risk factors, including hypercholesterolaemia which is associated with increased platelet activity. We examined platelet Abeta release under resting and collagen-stimulated conditions in normocholesterolaemic and hypercholesterolaemic individuals. Resting platelet Abeta efflux was greater in hypercholesterolaemics than in normocholesterolaemics. Collagen-stimulated Abeta release was concentration-dependent and increased in hypercholesterolaemics. Resting Abeta release correlated positively with plasma total cholesterol and low-density lipoprotein (LDL) cholesterol, and inversely with platelet count. These data indicate that abnormal platelet Abeta release occurs in hypercholesterolaemia.

By determining the sum of the supernatant concentrations of nitrite and nitrate the stimulated generation of nitric oxide (NO) by human washed platelets induced by a range of fibrillar collagen concentrations (0.0156-25 microg ml(-1)) was investigated. Platelet serotonin (5-hydroxytryptamine, 5-HT) efflux and platelet aggregation were also measured. Under resting conditions (0 microg ml(-1) collagen) platelet NO release was equivalent to 1.06+/-0.17 nmol per 10(8) platelets. Maximal NO release, equivalent to 2.1+/-0. 37 nmol per 10(8) platelets, was observed with only 0.0625 microg ml(-1) collagen (P

AIMS The aim of the study was to obtain further information regarding the modes of action of doxazosin, naftopidil and nifedipine on platelet function. METHODS We conducted an in vitro study of drug influences on adrenaline and collagen-induced mobilization of platelet calcium. RESULTS In the presence of fibrinogen (300 micrograms ml-1) both collagen (5 micrograms ml-1) and adrenaline (16 microM) stimulated the aggregation of washed platelets. Collagen induced a transient rise (+4.97 +/- 0.63 microM) in platelet Ca2+ concentration, [Ca2+]i, as measured using the photoprotein aequorin, which coincided with the onset of aggregation. Adrenaline induced a smaller rise (+3.6 +/- 0.96 microM) which, however, occurred after the onset of aggregation. Naftopidil, an alpha 1-adrenoreceptor antagonist produced a concentration-dependent inhibition of collagen-induced Ca2+ mobilization, maximum inhibition (22.9 +/- 4%, P < 0.05) occurring with 40 microM naftopidil. The inhibition of Ca2+ mobilization was not reflected by a concentration-dependent inhibition of platelet aggregation, although 40 microM naftopidil produced statistically significant inhibition (23.3 +/- 11.7%, P < 0.05). The adrenaline-induced rise in [Ca2+]i was inhibited dose dependently by naftopidil (e.g. 40 microM naftopidil, 100 +/- 0%, P < 0.05), as was aggregation (40 microM naftopidil, 100 +/- 0%, P < 0.05). Doxazosin, another alpha 1-adrenoreceptor blocker, inhibited Ca2+ mobilization induced by collagen to similar extents as for naftopidil (30 microM doxazosin, 17.4 +/- 2.5%, P < 0.05), but did not inhibit platelet aggregation. It also inhibited the adrenaline-induced rise in [Ca2+]i in a concentration-dependent manner (30 microM doxazosin, 37.6 +/- 13.7%, P < 0.05), significant inhibitions of platelet aggregation also being produced (30 microM, 49.6 +/- 17.2%, P < 0.05). As expected, the calcium channel blocker nifedipine produced concentration-dependent inhibitions of both collagen-induced Ca2+ mobilization (e.g. 28 microM nifedipine, 47.8 +/- 2.7%, P < 0.05) and aggregation (28 microM, 55.1 +/- 9.2%, P < 0.05). CONCLUSIONS These data indicate that the alpha 1-adrenoreceptor blockers, naftopidil and doxazosin, inhibit Ca2+ mobilization, this mechanism being possibly the means whereby these drugs inhibit platelet aggregation.

Collagen (5 microg/ml) stimulation of washed platelets increased endogenous serotonin (5-HT) release to the medium from 13.88 1.39 to 188.67 26.37 pmol/108 platelets ( P < 0.001). Adrenaline (16 microM) also increased 5-HT release, from 11.0 1.46 to 110.6 29.9 pmol/108 platelets ( P < 0.02). Naftopidil enhanced collagen-induced 5-HT efflux; significant increases occurred with 2 microM (+71.6%, P < 0.01), 10 microM (+89.1%, P < 0.01) and 40 microM (+69.7%, P < 0.01). With 0.4 muM and 2 microM naftopidil, adrenaline-induced 5-HT release was enhanced, albeit non-significantly, whilst with 10 microM and 40 muM naftopidil release was reduced (40 microM,-58.5%, P < 0.05). Doxazosin increased collagen-induced 5-HT release, significant increases being recorded with 7.5 microM (+81.7%, P < 0.05) and 30 microM (+78.4%, P < 0.05). Adrenaline-induced 5-HT release was also increased by doxazosin, but not significantly. Collagen-stimulated 5-HT release was inhibited by nifedipine (7 microM,-38.8%, P < 0.05; 28 microM, -61.2%, P < 0.001). These data suggest that the-antagonists, naftopidil and doxazosin, and the Ca2+ channel blocker, nifedipine, influence agonist-induced platelet 5-HT release through different mechanisms. Thus naftopidil and doxazosin may possess 5-HT transporter-blocking activity. The observation that naftopidil inhibited, adrenaline-induced 5-HT release may indicate that naftopidil also inhibits adrenaline uptake and exchange with dense granular 5-HT, with consequent inhibition of 5-HT release and platelet aggregation. The data obtained with nifedipine are consistent with 5-HT release being reduced as a result of its inhibitory action on platelet Ca2+ mobilisation.

1. Aggregation in platelet-rich plasma from normotensive men was induced by adrenaline (0.25-16 microM), ADP (0.25-16 microM), collagen (0.25-8 micrograms ml-1) or serotonin (10 microM) alone, or by previously sub-threshold concentrations of adrenaline (0.03-1 microM) in combination with sub-threshold concentrations of serotonin (2.5 microM), ADP (0.5 microM) or collagen (0.125 micrograms ml-1). The effects of the alpha 1-adrenoceptor blockers naftopidil and doxazosin on platelet aggregation were investigated. 2. The dose-response curves for collagen and ADP were unaffected by either drug. However, naftopidil (40 microM) inhibited serotonin-induced platelet aggregation (23.9%, 95% confidence interval (CI) 10.7 to 37.1%; P < 0.01) and caused a slight shift to the right of the adrenaline dose-response curve with a mean increase in the EC50 value of 0.5 microM (95% CI 0.07 to 0.93 microM; P < 0.05). Doxazosin had no effect on serotonin or adrenaline-induced aggregation. 3. A marked potentiation of the aggregation induced by subthreshold concentrations of adrenaline resulted from the prior addition of low concentrations of ADP, collagen or serotonin. 4. These potentiated responses were inhibited in a dose-dependent manner by naftopidil and to a lesser extent doxazosin. The maximum inhibitions (%) produced by naftopidil (40 microM) on the responses of adrenaline potentiated by ADP were 58.3% (95% CI 36.8 to 79.8%; P < 0.001), serotonin 58.9% (95% CI 40.0 to 77.8%; P < 0.001), and collagen 70.9% (95% CI 52.5 to 89.3%; P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Platelets and plasma lipoproteins, particularly low density lipoprotein, have important roles in atherogenesis. Evidence from several sources suggests that important interactions occur between these individual components of the atherogeneic process. Here we review work from our own laboratory on platelet function in normal individuals and patients heterozygous for familial hypercholesterolaemia (FH). Data is presented on the role of platelet noradrenaline and also on altered cellular signalling in platelets from FH individuals who have plasma low density lipoprotein concentrations which are approximately double those seen in normal subjects.

1. Plasma and platelet free catecholamine concentrations were measured in 22 normal subjects and in 10 treated and 11 untreated patients with heterozygous familial hypercholesterolaemia. 2. Plasma noradrenaline concentrations were significantly higher in both treated and untreated hypercholesterolaemic patients than in normal subjects. Adrenaline concentrations did not differ. 3. Platelet noradrenaline levels were higher in untreated hypercholesterolaemic patients than in normal subjects. 4. Positive correlations between the plasma noradrenaline concentration and the platelet noradrenaline concentration were observed in both normal subjects and hypercholesterolaemic patients. 5. Combining the data for normal subjects and hypercholesterolaemic patients revealed that the plasma noradrenaline concentration correlated positively with the plasma cholesterol concentration. The platelet noradrenaline concentration was also found to correlate with the plasma cholesterol concentration. 6. Our results suggest that an increased plasma cholesterol concentration may be associated with increased sympathetic nervous system activity as indicated by elevated plasma and platelet noradrenaline levels. Increases in circulating catecholamines may contribute to the platelet hyperaggregability seen in familial hypercholesterolaemia.

1. Energy expenditure, plasma glucose and palmitate kinetics and leg glycerol release were determined simultaneously both before and during adrenaline infusion in lean and obese human subjects. Seven lean subjects (mean 96.5% of ideal body weight) were studied in the post-absorptive state and also during mixed nutrient liquid feeding, eight obese subjects (mean 165% of ideal body weight) were studied in the post-absorptive state and six obese subjects (mean 174% of ideal body weight) were studied during feeding. 2. Resting energy expenditure was higher in the obese subjects, but the thermic response to adrenaline, both in absolute and percentage terms, was similar in lean and obese subjects. Plasma adrenaline concentrations attained (3 nmol/l) were comparable in all groups and the infusion had no differential effects on the plasma insulin concentration. Before adrenaline infusion the plasma glucose flux was higher in the obese than in the lean subjects in the fed state only (45.8 ± 3.8 versus 36.6 ± 1.0 mmol/h, P 3. Before the adrenaline infusion plasma palmitate flux was higher in the obese than in the lean subjects (by 51%, P 4. Before adrenaline infusion the palmitate oxidation rate was also higher in the obese than in the lean subjects (1.86 ± 0.14 versus 1.22 ± .09 mmol/h, P 5. These results suggest that in obese subjects plasma inter-organ transport of fatty acids and the subsequent fractional oxidation responses favour storage of triacylglycerol. These factors may be important determinants for the development and maintenance of the obese state.

1. In order to observe and compare the withdrawal phenomena which follow treatment with the beta-adrenoceptor blocking drugs, bopindolol (with partial agonist activity PAA) and atenolol (without PAA), two groups of six normal volunteers were studied before, during and after 16 days drug administration. 2. Measurements of plasma levels of cortisol, prolactin, insulin, noradrenaline, adrenaline, glucose and potassium were made during a pre-treatment baseline period, on maximum dose and for 21 days after drug withdrawal. Isoprenaline infusions were given to determine sensitivity of heart rate responses and haemodynamic changes measured in response to physiological manoeuvres. 3. Following atenolol withdrawal the results show hormonal evidence of adrenergic overactivity in the form of elevation of plasma cortisol, insulin and glucose levels. After bopindolol withdrawal there was, in contrast, an overshoot of plasma prolactin and a persistent elevation of plasma potassium and adrenaline post-isoprenaline. 4. The hormonal changes which follow withdrawal of atenolol and bopindolol are associated with haemodynamic changes reported elsewhere (Walden et al., 1990). 5. These observations provide confirmatory evidence of a post beta-adrenoceptor blockade withdrawal syndrome which differs between the two drugs studied and this may reflect the properties of the drugs, in particular the PAA of bopindolol.

1. Using high-performance liquid chromatography with electrochemical detection, we have studied the release of endogenous catecholamines from washed platelets induced to aggregate by ADP and thrombin. 2. Washed platelets exhibit irreversible aggregation with 10 μmol/l ADP and 0.3 unit of thrombin/ml, extents of aggregation being 27% and 76% respectively. 3. ADP (10μmol/l) increased noradrenaline release to the medium by 20% and adrenaline by 28%. As observed with aggregation, 0.3 unit of thrombin/ml produced a more marked effect on release than ADP, noradrenaline and adrenaline being increased by 570% and 169% respectively. 4. Values for platelet noradrenaline content were found to mirror those for the release from platelets induced by thrombin. 5. A correlation was observed between catecholamine release and the concentration of thrombin present in incubations. These data reflect the changes observed with platelet aggregation. 6. This is the first study to determine catecholamine release from platelets by direct measurement. Local catecholamine release after platelet aggregation in vivo may have important consequences for tissue perfusion.

1. Endogenous noradrenaline release from washed platelets incubated under resting conditions and in the presence of thrombin was examined in 14 normal subjects and 10 subjects with type 1 (insulin-dependent) diabetes. 2. Irreversible aggregation of platelets from both normal and diabetic subjects was induced by thrombin (0.3 unit/ml). Platelets from diabetic subjects were more sensitive than platelets from normal subjects, extents of aggregation being 89% and 76%, respectively (P < 0.002). 3. Stimulation with thrombin (0.3 unit/ml) elicited marked platelet release of noradrenaline to the incubation medium in both normal and diabetic subjects. Supernatant noradrenaline concentrations obtained under thrombin-stimulated conditions did not significantly differ between normal and diabetic subjects. However, under resting conditions noradrenaline levels were significantly greater (+ 93%, P < 0.02) for diabetic than normal subjects. 4. Measurement of platelet noradrenaline contents after thrombin stimulation revealed no difference between normal and diabetic subjects. Under resting conditions, however, platelet noradrenaline levels were significantly lower (−46%, P < 0.02) for diabetic than normal subjects. Thus, in the diabetic subjects increased resting platelet efflux of noradrenaline is mirrored by a decreased platelet noradrenaline content. 5. A consequence of increases in resting catecholamine efflux may be enhanced platelet activity resulting in increased platelet aggregation.

1. We have used high-performance liquid chromatography with electrochemical detection to measure plasma and platelet catecholamines in 24 normal subjects. 2. In the same subjects platelet function was assessed by measuring platelet aggregation in response to adenosine 5′-pyrophosphate, thrombin, adrenaline and collagen. Platelet sensitivity to prostacyclin was also examined. 3. Platelet noradrenaline showed a positive correlation with extent of aggregation induced by ‘low-dose’ collagen (1 μg/ml). No correlation was seen at the higher collagen concentration. 4. Platelet noradrenaline content also correlated with sensitivity of platelets to prostacyclin. High platelet noradrenaline concentrations appeared to result in decreased sensitivity to prostacyclin. 5. No other correlations were observed. 6. These data suggest that platelet noradrenaline rather than plasma levels may be involved in modifying platelet function in vivo. Local release of platelet catecholamines may affect the platelet/vessel wall interaction, the primary physiological step in platelet activation.

We have examined resting and thrombin (0.3 units ml-1) induced release of noradrenaline by washed platelets from 15 normal subjects and eight patients with heterozygous familial hypercholesterolaemia. Platelets from both normal and hypercholesterolaemic subjects showed irreversible aggregation with 0.3 units ml-1 thrombin. Extents of aggregation were 76.3% and 90.8% respectively, platelets from hypercholesterolaemic patients being significantly more sensitive (P less than 0.002). Under resting conditions platelet noradrenaline release was 136% greater (P less than 0.02) in hypercholesterolaemic patients than in normal subjects. Thrombin-stimulated release of noradrenaline was also higher (73%, P less than 0.05) in hypercholesterolaemics than in normals. The differences between resting and thrombin-stimulated release were greater for hypercholesterolaemic patients than normal subjects (P less than 0.05). Under resting conditions total platelet noradrenaline levels (sum of supernatant and platelet pellet concentrations) were similar in preparations from the two groups. However, following thrombin stimulation total noradrenaline concentrations were substantially greater (86%) in platelets from hypercholesterolaemics than normals (P less than 0.02). In hypercholesterolaemic patients thrombin stimulation was associated with an 101% increase (over resting levels) in total platelet noradrenaline (P less than 0.01), no increases being observed with normal subjects. We suggest that platelet membranes may be more permeable in patients with familial hypercholesterolaemia leading to increased non-specific release of catecholamines. Platelets from patients with familial hypercholesterolaemia may also be more responsive to stimulation with respect to catecholamine release. The results obtained on calculation of total platelet noradrenaline levels may indicate that abnormalities of platelet dense granules occur in familial hypercholesterolaemia. In this context the relative proportions of free and conjugated catecholamine may be of relevance.

The effects of the irreversible inhibitors of GABA-transaminase (E.C.2.6.1.19), γ-acetylenic GABA (4-amino-hex-5-ynoic acid, RMI 71645) and γ-vinyl GABA (4-amino-hex-5-enoic acid, RMI 71754) on the release and uptake of endogenous and exogenous GABA by rat cerebral cortical synaptosomes were studied. γ-Acetylenic GABA increased the release of preloaded [U 14 C]-GABA by 12 to 46% over the concentration range 0.5 to 5 mM. With γ-vinyl GABA, the concentration range 0.25–5 mM caused an increase of 22–95% above controls. All concentrations of γ-vinyl GABA were more effective than equivalent levels of γ-acetylenic GABA. The two agents had similar effects on the release of endogenous GABA, no change was observed in any other amino acid. The effect of both drugs on [U 14 C]-GABA release was inhibited significantly by tetrodotoxin and blocked by Verapamil. When animals were pretreated with γ-acetylenic GABA (100 mg/kg) for 8 hr or γ-vinyl GABA (1000 mg/kg) for 14 hr, the control release of GABA from synaptosomes prepared subsequently was increased, but other amino acids showed no change. The GABA content of the tissue was also greatly increased. The veratrine-stimulated release of GABA, glutamate and aspartate were increased by this pretreatment, but no change occurred in the rates of release of other amino acids. All veratrine-stimulated changes in the release of endogenous GABA, glutamate and aspartate were prevented by tetrodotoxin.