The rare C9 P167S risk variant for age-related macular degeneration increases polymerization of the terminal component of the complement cascade.

Age-related macular degeneration (AMD) is a complex neurodegenerative eye disease with behavioral and genetic etiology and is the leading cause of irreversible vision loss among elderly Caucasians. Functionally significant genetic variants in the alternative pathway of complement have been strongly linked to disease. More recently, a rare variant in the terminal pathway of complement has been associated with increased risk, Complement component 9 (C9) P167S. To assess the functional consequence of this variant, C9 levels were measured in two independent cohorts of AMD patients. In both cohorts, it was demonstrated that the P167S variant was associated with low C9 plasma levels. Further analysis showed that patients with advanced AMD had elevated sC5b-9 compared to those with non-advanced AMD, although this was not associated with the P167S polymorphism. Electron microscopy of membrane attack complexes (MACs) generated using recombinantly produced wild type or P167S C9 demonstrated identical MAC ring structures. In functional assays, the P167S variant displayed a higher propensity to polymerize and a small increase in its ability to induce hemolysis of sheep erythrocytes when added to C9-depleted serum. The demonstration that this C9 P167S AMD risk polymorphism displays increased polymerization and functional activity provides a rationale for the gene therapy trials of sCD59 to inhibit the terminal pathway of complement in AMD that are underway.

Complement factor I: Regulatory nexus, driver of immunopathology, and therapeutic.

Complement factor I (FI) is the nexus for classical, lectin and alternative pathway complement regulation. FI is an 88 kDa plasma protein that circulates in an inactive configuration until it forms a trimolecular complex with its cofactor and substrate whereupon a structural reorganization allows the catalytic triad to cleave its substrates, C3b and C4b. In keeping with its role as the master complement regulatory enzyme, deficiency has been linked to immunopathology. In the setting of complete FI deficiency, a consumptive C3 deficiency results in recurrent infections with encapsulated microorganisms. Aseptic cerebral inflammation and vasculitic presentations are also less commonly observed. Heterozygous mutations in the factor I gene (CFI) have been demonstrated to be enriched in atypical haemolytic uraemic syndrome, albeit with a very low penetrance. Haploinsufficiency of CFI has also been associated with decreased retinal thickness and is a strong risk factor for the development of age-related macular degeneration. Supplementation of FI using plasma purified or recombinant protein has long been postulated, however, technical difficulties prevented progression into clinical trials. It is only using gene therapy that CFI supplementation has reached the clinic with GT005 in phase I/II clinical trials for geographic atrophy.

A scanning Kelvin probe study of charge trapping in zone-cast pentacene thin film transistors.

We have used scanning Kelvin probe microscopy (SKPM) as a local probe to study charge trapping in zone-cast pentacene field effect transistors on both SiO(2) and benzocyclobutene (BCB) substrates. Annealing at 130 degrees C was found to reduce the threshold voltage, susceptibility to negative gate bias stress and trapping of positive charges within single pentacene grains. We conclude that oxygen is able to penetrate and disassociatively incorporate into crystalline pentacene, chemically creating electrically active defect states. Screening of a positive gate bias caused by electron injection from Au into pentacene was directly observed with SKPM. The rate of screening was found to change significantly after annealing of the film and depended on the choice of gate dielectric.

Protein kinase C: is its pivotal role in cellular activation over-stated?

Evidence has emerged over the past decade to suggest that protein kinase C (PKC) is a widespread family of kinases responsible for many diverse and critical cellular functions. With the development of selective agents to activate or inhibit the individual PKC isoenzymes, it is now apparent that much of the literature that implicated PKC in many cellular functions needs to be appraised. In this article, Sandra Wilkinson and Trevor Hallam discuss the problems of the existing methods and the recent evidence that suggests that PKC isotypes are necessary for some, but not all, of those cellular responses where PKC had been thought to play an important role. Selective inhibitors of PKC isoenzymes may have potential for therapeutic use in auto-immune diseases, transplant rejection and oncology.

The involvement of calcium and protein kinase C in modulating agonist-stimulated chemotaxis of human neutrophils.

Chemotaxis of human neutrophils in response to a gradient of the chemotactic peptide, fmet-leu-phe (FMLP), was measured by the under-agarose technique. The dose-response curve for FMLP was biphasic; low concentrations were stimulatory, and the response was reduced at higher concentrations. The response to FMLP was partially inhibited (about 50%) in the absence of extracellular Ca2 (EGTA added). NiCl2 dose-dependently inhibited FMLP-stimulated chemotaxis in the presence of extracellular Ca2+; the maximum inhibition obtainable with NiCl2 was similar to that with the absence of extracellular Ca2+. These results suggest that FMLP-stimulated chemotaxis is, at least partially, dependent on stimulation of Ca2+ influx. The phorbol ester, PMA, dose-dependently inhibited chemotaxis; the response was almost completely inhibited by 10 nM PMA. This result indicates that activation of protein kinase C inhibits chemotaxis. These results are discussed in relation to the physiological responses of neutrophils.

Calcium signalling in non-excitable cells: notes on oscillations and store refilling.

Technical advances in studying cellular calcium concentrations, and discoveries about many aspects of signal transduction have transformed this field of biology since this Journal was launched a decade ago. At that time monitoring of the key variable, cytoplasmic free Ca2+ concentration [Ca2+]i, was possible with aequorin or arsenazo ill mainly in large invertebrate cells, though pioneering work with aequorin micro-injection into cardiac and smooth muscle had just started. At that time there was also intense activity by a few groups aiming to make Ca-selective micro-electrodes selective and sharp enough to measure [Ca2+]i in small cells. Also the use of electropermeabilized cells had begun to allow the defining of the concentrations of Ca2+ required to activate secretion in mammalian cells. Nearly all this work and all the relevant electrophysiology relating to calcium signalling had been done in excitable cells, basically muscle and nerve, and was aimed at understanding contraction, transmitter release and neurosecretion, and the control of membrane permeability. Recent advances have now allowed [Ca2+]i to be measured in non-excitable cells.

Agonists stimulate divalent cation channels in the plasma membrane of human platelets.

Agonists such as thrombin, PAF (platelet-activating factor) and ADP are known to cause a larger elevation in [Ca2+]i in quin2-loaded platelets in the presence of extracellular Ca2+ than in its absence. The simplest interpretation of these observations is that in the presence of extracellular calcium there is an influx component across the cell surface. In the presence of Mn2+, a divalent cation which is known to avidly bind to quin2 and to quench its fluorescence, the agonists produce a small initial rise in quin2 fluorescence followed by a decrease in fluorescence to well below the resting level. The result indicates entry of Mn2+, presumably through some form of receptor-operated Ca2+ channel.

Exogenous ATP raises cytoplasmic free calcium in fura-2 loaded piglet aortic endothelial cells.

Cultured piglet endothelial cells were grown to confluence on glass coverslips and loaded with the fluorescent Ca2+ indicator, fura-2. Using a dual-wavelength excitation fluorescence spectrophotometer it was found that ATP caused a rapid transient elevation in [Ca2+]i in the presence of extracellular calcium which decreased to a maintained elevated level. With no extracellular calcium ATP evoked a similar transient increase which returned to the basal level. Addition of 50 mM K+ had no effect on [Ca2+]i or on the effect of ATP on [Ca2+]i in the presence of extracellular Ca2+. The data suggest that ATP causes both discharge of calcium from an intracellular pool and influx across the plasma membrane although this is unlikely to be via a voltage-operated channel. ATP stimulated simultaneously the production of PGI2 to a similar extent in the presence or absence of extracellular calcium. Elevated [Ca2+]i may be an important activation pathway in the endothelial cell.

Trifluoperazine and chlorpromazine block secretion from human platelets evoked at basal cytoplasmic free calcium by activators of C-kinase.

Trifluoperazine, chlorpromazine and other drugs known to inhibit calmodulin-dependent processes are also known to inhibit protein kinase C. The effect of these agents on secretion evoked by known activators of C-kinase has been studied in human platelets loaded with the fluorescent Ca indicator, quin2 and preincubated with aspirin. The secretory response stimulated by phorbol ester and exogenous diacylglycerol, at basal levels of cytoplasmic free Ca2+, [Ca2+]i, was suppressed by trifluoperazine, chlorpromazine and W-7, as was the secretion evoked by collagen that occurs without a change in [Ca2+]i. The response to thrombin, which is accompanied by elevated [Ca2+]i was barely affected. Modest elevation of [Ca2+]i by Ca ionophore was able to overcome the inhibitory effect of these drugs on the response to phorbol ester, diacylglycerol, and collagen.

Low concentrations of the stable prostaglandin endoperoxide U44069 stimulate shape change in quin2-loaded platelets without a measurable increase in [Ca2+]i.

Dose-response relationships for raised cytoplasmic free calcium concentration, [Ca2+]i, and shape change were measured simultaneously in quin2-loaded human platelets. With the calcium ionophore ionomycin the threshold [Ca2+]i for shape change was 300 nM with a maximal response at 800 nM. With 1 mM external Ca2+ the U44069 concentrations required to stimulate half-maximal shape change and an increase in [Ca2+]i were 2 and 41 nM, respectively. For PAF these values were 8.7 and 164 pg/ml, respectively. Low concentrations of U44069 and PAF evoked substantial shape change without any rise in [Ca2+]i. In the absence of external Ca2+, U44069 stimulated half-maximal shape change at 2 nM, and half-maximal elevation of [Ca2+]i at 69 nM: here, increased [Ca2+]i never reached the threshold [Ca2+]i for shape-change derived with ionomycin. These results suggest that some transduction mechanism other than elevated [Ca2+]i, as yet unidentified, can cause shape change.

The role of 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (AcGEPC) and palmitoyl-lysophosphatidate in the responses of human blood platelets to collagen and thrombin.

Desensitisation of human blood platelets to the effects of 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (1-O-alkylAcGEPC) and palmityl-lysophosphatidate by pre-incubation with these agonists has no effect on the aggregatory or secretory responses to collagen but causes 30-40% inhibition of these responses to thrombin in aspirin-treated platelets. The effects of 1-O-alkylAcGEPC and palmitoyl-lysophosphatidate are not additive. The results are not consistent with the proposal that 1-O-alkylAcGEPC or lysophosphatidate are the mediators for the responses to collagen observed when prostaglandinendoperoxide synthesis is prevented, although they may play some role in the responses to thrombin under these conditions.

TMB-8 inhibits secretion evoked by phorbol ester at basal cytoplasmic free calcium in quin2-loaded platelets much more effectively than it inhibits thrombin-induced calcium mobilisation.

TMB-8 is widely regarded as an 'intracellular calcium antagonist', supposedly inhibiting the mobilisation of intracellular calcium. Rarely, however, have the effects of this compound on Ca2+ movements been measured. We report here that TMB-8 is not very effective in inhibiting thrombin-induced Ca2+ influx or internal release in human platelets judged from the fluorescent signal of cytoplasmic quin2. Only approx. 40% inhibition was seen at 500 microns TMB-8. Somewhat lower concentrations blocked the secretory response to thrombin and also the secretion evoked at basal [Ca2+]i by phorbol ester and collagen. It is suggested that one target for TMB-8 may be the C-kinase pathway.

Regulation of P2y-purinoceptor-mediated prostacyclin release from human endothelial cells by cytoplasmic calcium concentration.

1. ATP and ATP analogues induced prostacyclin (PGI2) secretion from human cultured umbilical vein endothelial cells. 2. The threshold active concentration for ATP was less than or equal to 1 microM. The rank order of potency of analogues was 2-chloroadenosine 5'-triphosphate (2-ClATP) greater than 2-methylthioadenosine 5'-triphosphate (2-MeSATP) greater than ATP greater than ADP, while adenosine 5'-(alpha,beta-methylene)triphosphonate, AMP and adenosine were inactive, indicating the presence of P2y-purinoceptors. 3. In contrast to their actions on P2y-receptors in guinea-pig taenia coli, isopolar analogues of 2-methylthioadenosine 5'-(beta, gamma-methylene)triphosphonate were less effective than ATP. 4. ATP and ATP analogues increased intracellular free calcium ions, [Ca2+]i, giving a rapid transient peak due predominantly to release from intracellular stores, followed by a maintained steady-state elevated level due to influx. 5. The dose-response curves for peak [Ca2+]i induced by ATP, 2-ClATP and 2-MeSATP were very similar to those for PGI2 production. 6. Elevations of [Ca2+]i, above a threshold value of 0.8-1 microM, were necessary for PGI2 production in response to P2y-receptor activation. 7. The dose relationships between PGI2 release and peak [Ca2+]i were equivalent whether [Ca2+]i was raised by ionomycin or via P2y-receptor activation by ATP or 2-ClATP, indicating that elevations of [Ca2+]i provide the major, if not the exclusive intracellular pathway for P2y-purinoceptor-mediated PGI2 synthesis.

The effects of siguazodan, a selective phosphodiesterase inhibitor, on human platelet function.

1. The effects of siguazodan (SK&F 94836) a selective phosphodiesterase (PDE) inhibitor with inotropic and vasodilator activity, were studied on human platelets. 2. Siguazodan selectively inhibited the major cyclic AMP-hydrolysing PDE in human platelet supernatants. The inhibited enzyme has been variously termed cyclic GMP-inhibited PDE or PDE-III. 3. In platelet-rich plasma (PRP), siguazodan inhibited U46619-induced aggregation more potently than that induced by ADP and collagen. Treatment of the PRP with aspirin had no effect on the potency of siguazodan. 4. In washed platelets, siguazodan increased cyclic AMP levels and reduced cytoplasmic free calcium [( Ca2+]i). ADP decreased the ability of siguazodan to raise cyclic AMP and this may explain its lower potency in inhibiting responses to ADP. 5. Siguazodan has anti-platelet actions over the same concentration range that it is an inotrope and vasodilator.

Octimibate, a potent non-prostanoid inhibitor of platelet aggregation, acts via the prostacyclin receptor.

1. Octimibate, 8-[(1,4,5-triphenyl-1H-imidazol-2-yl)oxy]octanoic acid, is reported to have antithrombotic properties. This is in addition to its antihyperlipidaemic effects which are due to inhibition of acylCoA:cholesterol acyltransferase (ACAT). The aim of this study was to investigate the mechanism of the antithrombotic effect of octimibate, and to determine whether the effects of octimibate are mediated through prostacyclin receptors. 2. In suspensions of washed (plasma-free) human platelets, octimibate is a potent inhibitor of aggregation; its IC50 is approx. 10 nM for inhibition of aggregation stimulated by several different agonists, including U46619 and ADP. The inhibitory effects of octimibate on aggregation are not competitive with the stimulatory agonist; the maximal response is suppressed but there is no obvious shift in potency of the agonist. In platelet-rich plasma, octimibate inhibits agonist-stimulated aggregation with an IC50 of approx. 200 nM. 3. Octimibate also inhibits agonist-stimulated rises in the cytosolic free calcium concentration, [Ca2+]i, in platelets. Both Ca2+ influx and release from intracellular stores are inhibited. The effects of octimibate on aggregation and [Ca2+]i are typical of agents that act via elevation of adenosine 3':5'-cyclic monophosphate (cyclic AMP). Similar effects are seen with forskolin, prostacyclin (PGl2) and iloprost (a stable PGl2 mimetic). 4. Octimibate increases cyclic AMP concentrations in platelets and increases the cyclic AMP-dependent protein kinase activity ratio. Octimibate stimulates adenylyl cyclase activity in human platelet membranes, with an EC50 of 200 nM. The maximal achievable activation of adenylyl cyclase by octimibate is 60% of that obtainable with iloprost. Octimibate has no effect on the cyclic GMP-inhibited phosphodiesterase (phosphodiesterase-ITI), which is the major cyclic AMP-degrading enzyme in human platelets.5. Octimibate inhibits, apparently competitively, the binding of [3H]-iloprost (a stable PGl2 mimetic) to platelet membranes; the estimated Ki is 150 nm. 6. The platelets of different species show considerable differences in the apparent potency of their inhibition of aggregation by octimibate; platelets from cynomolgus monkeys are 3 fold more sensitive than those from humans, while rat, cat and cow platelets are 50, 100, and 250 fold less sensitive than human platelets. The sensitivity of these different species to iloprost, however, varies over a range of only 10 fold with no obvious difference between primates and non-primates. 7. Octimibate appears to be a potent agonist (aggregation), or partial agonist (adenylyl cyclase), at prostacyclin receptors and is the first non-prostanoid agent of this type to be identified. The species differences in relative potency of octimibate and iloprost may reflect the existence of receptor subtypes.

Primate vascular responses to octimibate, a non-prostanoid agonist at the prostacyclin receptor.

1. Octimibate is a potent inhibitor of human platelet aggregation, and appears to act (at least in part) through the prostacyclin receptor, as described in the preceding paper. Here, the vascular effects, both in vitro and in vivo, of octimibate have been compared to those of the stable prostacyclin (PGI2) mimetic, iloprost. Since octimibate shows extensive species variation and is potent at inhibiting platelet aggregation in primates, all of the experiments reported here have been carried out with primate tissue or in vivo in cynomolgus monkeys. 2. Activation of adenylyl cyclase in human lung membranes appears to involve stimulation of the vascular PGI2 receptor. Octimibate, as well as iloprost, stimulates adenylyl cyclase in this preparation. The EC50 values for iloprost and octimibate are 50 nM and 340 nM respectively. These values are similar to those seen with human platelet membranes. As with platelets, the maximal activation achievable with octimibate is 60% of that seen with iloprost. This result suggests that octimibate is a partial agonist for stimulation of adenylyl cyclase. 3. Iloprost (10-100 nM) relaxes human coronary and mesenteric artery precontracted with KCl, and also relaxes cynomolgus monkey aorta precontracted with phenylephrine. Octimibate appears to be a partial agonist for relaxation of human coronary artery precontracted with KCl; the intrinsic activity of octimibate (10 microM) is 0.15 compared to iloprost, and octimibate surmountably antagonizes the relaxant effects of iloprost with a Kp of 200 nM. Octimibate (up to 10 microM) evokes only weak relaxation of human mesenteric artery (precontracted with KCl) and cynomolgus monkey aorta (precontracted with phenylephrine). 4. The effects of iloprost and octimibate were compared in vivo in cynomolgus monkeys. In addition to inhibiting ex vivo platelet aggregation, both compounds cause hypotension with little effect on heart rate. The dose-response curves for inhibition of ex vivo platelet aggregation and a fall in mean arterial blood pressure were compared. The dose-separation (i.e., the relative differences in effective concentrations) for the two responses is similar with both iloprost and octimibate. 5. Since the pern; beral resistance vessels are intimately involved in regulation of systemic arterial blood pressure, the effects of both agents were tested on human peripheral resistance vessels (150-400pm diameter) in vitro. These vessels are relaxed by both iloprost and octimibate following precontraction with KCI. The IC50 value for iloprost is 44nM, and 1.7 microM octimibate evokes 50% of the maximal relaxation obtained with iloprost. Thus, the relative potencies of the two compounds in relaxing human subcutaneous resistance vessels are similar to their relative potencies in inhibiting platelet responses. This result correlates with the lack of platelet versus vascular selectivity seen with the in vivo monkey studies. 6. These results suggest that octimibate, a partial agonist at the prostacyclin receptor, is unable to discriminate between platelet and vascular prostacyclin receptors in primates.

Synergistic responses and receptor occupancy in rabbit blood platelets.

Several observations indicate that simultaneous receptor occupancy is necessary for generation of a synergistic response of rabbit platelets to two excitatory agonists. First, an aggregatory response to adrenaline induced by prior addition of 5HT reverses rapidly unless stabilised by inhibition of 5HT uptake. The extent of response correlates with the extracellular 5HT concentration. Second, addition of an ADPase following adrenaline enhances the rate of disaggregation if the response to this agonist has been induced by prior addition of ADP. Third, disaggregation is induced, or enhanced, if an antagonist selective to the inducing agonist is added following completion of the induced aggregatory response. These data suggest marked lability in the mechanisms responsible for generation of synergistic responses.

Changes in protein kinase C subspecies protein expression and activity in a series of multidrug-resistant human KB carcinoma cell lines.

We have investigated the relationship between protein kinase C (PKC), levels of resistance and drug used for selection in a series of human KB carcinoma cell lines by comparing protein kinase C activity and PKC alpha, beta I, beta II, gamma, delta, epsilon, and zeta subspecies protein expression. PKC alpha protein expression was increased by 600% and 375% in KB-A1 and KB-C1 lines respectively over the parent KB-3-1 line; only KB-A1 cells showed increased PKC delta expression. Expression of other PKC subspecies was equal to that of KB-3-1 cells. There was considerable variation between the different KB cell lines in total cytosolic PKC activity, the KB-A1 and KB-C1 lines showing 400% and 350% increases respectively, KB-V1 and KB-8-5-11 about 180%, and KB-8-5 no increase relative to the parent KB-3-1 line. For calcium-independent PKC activity, the KB-C1 and KB-A1 lines only were increased over the KB-3-1 line. Immunoprecipitation with antisera to PKC subspecies confirmed that the increase in KB-A1 cytosolic total PKC activity was due largely to PKC alpha and partially to PKC delta. Membrane-associated PKC activity was increased by 500% and 350% in KB-A1 and KB-C1 lines respectively, by 250% and 270% in KB-V1 and KB-8-5-11, and not increased in KB-8-5 cells relative to the KB-3-1 cells. For KB-C1, KB-8-5-11, and KB-8-5 lines, which show decreasing resistance to colchicine, our results suggest a correlation between PKC and multidrug resistance in cells selected for resistance to this drug. There is no correlation between PKC and multidrug resistance for cells selected in different drugs. Our study therefore suggests that specific PKC subspecies are associated with the MDR phenotype of some KB cell lines, but that the extent of PKC involvement depends on the type of drug used for selection and its concentration.

Functional significance of protein kinase C in human T-cell activation: a new therapeutic class?

The assumption that protein kinase C activation is necessary for T-cell activation in response to MHC class II restricted antigen-presentation is supported by several lines of circumstantial evidence. We have developed potent, cell-permeable and selective inhibitors of PKC to test this hypothesis. Our data demonstrate the crucial role for PKC in T-cell activation and that selective orally bioavailable PKC inhibitors are efficacious in preventing T-cell driven chronic inflammatory responses in vivo.

Compensation and stability in nonlinear matrix models.

Stability, bifurcation, and dynamic behavior, investigated here in discrete, nonlinear, age-structured models, can be complex; however, restrictions imposed by compensatory mechanisms can limit the behavioral spectrum of a dynamic system. These limitations in transitional behavior of compensatory models are a focal point of this article. Although there is a tendency for compensatory models to be stable, we demonstrate that stability in compensatory systems does not always occur; for example, equilibria arising through a bifurcation can be initially unstable. Results concerning existence and uniqueness of equilibria, stability of the equilibria, and boundedness of solutions suggest that "compensatory" systems might not be compensatory in the literal sense.