The effect of cis-5,8,11,14,17-eicosapentaenoic acid upon the contractile mechanisms linked to calcium influx and the mobilisation of intracellular calcium in aortic smooth muscle

Epidemiological studies previously identified cis-5,8,11,14,17-eicosapentaenoic acid (EPA) as the biologically active component of fish oil of benefit to the cardiovascular system. Although clinical investigations demonstrated its usefulness in surgical procedures, its mechanism of action still remained unclear. It was shown in this thesis, that EPA partially blocked the contraction of aortic smooth muscle cells to the vasoactive agents KCl and noradrenaline. The latter effect was likely caused by reducing calcium influx through receptor-operated channels, supporting a recent suggestion by Asano et al (1997). Consistently, EPA decreased noradrenaline-induced contractures in aortic tissue, in support of previous reports (Engler, 1992b). The observed effect of EPA on cell contractions to KCl was not simple due to blocking calcium influx through L-type channels, consistent with a previous suggestion by Hallaq et al (1992). Moreover, EPA caused a transient increase in [Ca2+]i in the absence of extracellular calcium. To resolve this it was shown that EPA increased inositol phosphate formation which, it is suggested, caused the release of calcium from an inositol phosphate-dependent internal binding site, possibly that of an intracellular membrane or superficial sarcoplasmic reticulum, producing the transient increase in [Ca2+]i. As it was shown that the cellular contractile filaments were not desensitised to calcium by EPA, it is suggested that the transient increase in [Ca2+]i subsequently blocks further cell contraction to KCl by activating membrane-associated potassium channels. Activation of potassium channels induces the cellular efflux of potassium ions, thereby hyperpolarising the plasma membrane and moving the membrane potential farther from the activation range for calcium channels. This would prevent calcium influx in the longer term and could explain the initial observed effect of EPA to block cell contraction to KCl.

The impact of the CACNA1C gene polymorphism on frontolimbic function in bipolar disorder

Genome-wide association studies in bipolar disorder (BD)1 have implicated a single-nucleotide polymorphism (rs1006737, G right arrow A) in the CACNA1C gene, which encodes for the alpha 1c (CAV1.2) subunit of the voltage-gated, L-type calcium channel. Neuroimaging studies of healthy individuals report that this risk allele modulates brain function within limbic (amygdala, anterior cingulate gyrus) and hippocampal regions during tasks of reward processing2, 3 and episodic memory. Moreover, animal studies suggest that the CaV1.2 L-type calcium channels influence emotional behaviour through enhanced neurotransmission via the lateral amygdala pathway. On the basis of this evidence, we tested the hypotheses that the CACNA1C rs1006737 risk allele will modulate neural responses within predefined prefrontal and subcortical regions of interest during emotional face processing and that this effect would be amplified in BD patients.

Differential control of two forms of glutamate release by group III metabotropic glutamate receptors at rat entorhinal synapses

Neurotransmitter release at CNS synapses occurs via both action potential-dependent and independent mechanisms, and it has generally been accepted that these two forms of release are regulated in parallel. We examined the effects of activation of group III metabotropic glutamate receptors (mGluRs) on stimulus-evoked and spontaneous glutamate release onto entorhinal cortical neurones in rats, and found a differential regulation of action potential-dependent and independent forms of release. Activation of presynaptic mGluRs depressed the amplitude of stimulus-evoked excitatory postsynaptic currents, but concurrently enhanced the frequency of spontaneous excitatory currents. Moreover, these differential effects on glutamate release were mediated by pharmacologically separable mechanisms. Application of the specific activator of adenylyl cyclase, forskolin, mimicked the effect of mGluR activation on spontaneous, but not evoked release, and inhibition of adenylyl cyclase with 9-tetrahydro-2-furanyl)-9H-purin-6-amine (SQ22536) blocked mGluR-mediated enhancement of spontaneous release, but not depression of evoked release. Occlusion studies with calcium channel blockers suggested that the group III mGluRs might depress evoked release through inhibition of both N and P/Q, but not R-type calcium channels. We suggest that the concurrent depression of action potential-evoked, and enhancement of action potential-independent glutamate release operate through discrete second messenger/effector systems at excitatory entorhinal terminals in rat brain. © 2007 IBRO.

Mouse splenocyte proliferation and its modulation by sex steroids

Concanavalin A, a T cell mitogen enhanced DNA synthesis in murine splenocytes. Amongst the early signals prior to this event was an increase in cytosolic calcium derived from both intra- and extracellular sources. The requirements for extracellular calcium persisted for four hours after the lectin administration which itself was needed for six hours. Putative calcium channel antagonists and calmodulin inhibitors blocked ihe increase in DNA synthesis. The calcium signal was mimicked by application of the ionophore, A23187, although no increase in DNA synthesis occurred. An activator of protein kinase C, 12-0- tetradecanoylphorbol 13-acetate, had little effect in isolation but the combined application of these two agents greatly enhanced DNA synthesis. The natural mediators of these events are presumed to be inositol trisphosphate and diacylglycerol derived from phosphatidylinositol bisphosphate hydrolysis. Lectin application and protein kinase C activation both increased intracellular pH possibly as a result of Na'l'/H"'' exchange since amiloride an inhibitor of this antiporter inhibited lectin induced DNA synthesis. The calcium and hydrogen ionic changes occur within minutes of lectin application; the protracted requirement for this mitogen suggests further signalling mechanisms occur to elicit maximum DNA synthesis in these cells. Gonadectomy caused an increase in thymic and splenic weight. Spleno-cytes derived from castrated mice showed no change in mitogen response whereas those from ovariectomised mice demonstrated a reduced lectin sensitivity. Testosterone, 5 a dihydrotestosterone, a and 0 oestradiol all inhibited lectin induced DNA synthesis but only at pharmacological concentrations. Testosterone glucuronide and cholesterol were without effect Studies with mouse serum fractions of differing steroidal status were unable to confirm the presence or absence of serum factors which might mediate the effects of steroid on lymphoid cells, all fractions tested inhibited lymphocyte transformation. Both interleukin-2 and lipopolysaccharide induced splenocyte mitogene-sis was also impaired by high steroid concentrations in vitro, suggesting that steroids mediate their effect by a non-specific, non-receptor-mediated event.

Genetic risk factors and age-related macular degeneration (AMD)

Age related macular degeneration (AMD) is the leading cause of blindness in individuals older than 65 years of age. It is a multifactorial disorder and identification of risk factors enables individuals to make lifestyle choices that may reduce the risk of disease. Collaboration between geneticists, ophthalmologists, and optometrists suggests that genetic risk factors play a more significant role in AMD than previously thought. The most important genes are associated with immune system modulation and the complement system, e.g., complement factor H (CFH), factor B (CFB), factor C3, and serpin peptidase inhibitor (SERPING1). Genes associated with membrane transport, e.g., ATP-binding cassette protein (ABCR) and voltage-dependent calcium channel gamma 3 (CACNG3), the vascular system, e.g., fibroblast growth factor 2 (FGF2), fibulin-5, lysyl oxidase-like gene (LOXL1) and selectin-P (SELP), and with lipid metabolism, e.g., apolipoprotein E (APOE) and hepatic lipase (LIPC) have also been implicated. In addition, several other genes exhibit some statistical association with AMD, e.g., age-related maculopathy susceptibility protein 2 (ARMS2) and DNA excision repair protein gene (ERCC6) but more research is needed to establish their significance. Modifiable risk factors for AMD should be discussed with patients whose lifestyle and/or family history place them in an increased risk category. Furthermore, calculation of AMD risk using current models should be recommended as a tool for patient education. It is likely that AMD management in future will be increasingly influenced by assessment of genetic risk as such screening methods become more widely available. © 2013 Spanish General Council of Optometry.

Self-association of calcium-binding protein S100A4 and metastasis

Elevated levels of the calcium-binding protein S100A4 promote metastasis and in carcinoma cells are associated with reduced survival of cancer patients. S100A4 interacts with target proteins that affect a number of activities associated with metastatic cells. However, it is not known how many of these interactions are required for S100A4-promoted metastasis, thus hampering the design of specific inhibitors of S100A4-induced metastasis. Intracellular S100A4 exists as a homodimer through previously identified, well conserved, predominantly hydrophobic key contacts between the subunits. Here it is shown that mutating just one key residue, phenylalanine 72, to alanine is sufficient to reduce the metastasis-promoting activity of S100A4 to 50% that of the wild type protein, and just 2 or 3 specific mutations reduces the metastasis-promoting activity of S100A4 to less than 20% that of the wild type protein. These mutations inhibit the self-association of S100A4 in vivo and reduce markedly the affinity of S100A4 for at least two of its protein targets, a recombinant fragment of non-muscle myosin heavy chain isoform A, and p53. Inhibition of the self-association of S100 proteins might be a novel means of inhibiting their metastasis-promoting activities.

Analysis of the pore of the unusual major intrinsic protein channel, yeast Fps1p

Fps1p is a glycerol efflux channel from Saccharomyces cerevisiae. In this atypical major intrinsic protein neither of the signature NPA motifs of the family, which are part of the pore, is preserved. To understand the functional consequences of this feature, we analyzed the pseudo-NPA motifs of Fps1p by site-directed mutagenesis and assayed the resultant mutant proteins in vivo. In addition, we took advantage of the fact that the closest bacterial homolog of Fps1p, Escherichia coli GlpF, can be functionally expressed in yeast, thus enabling the analysis in yeast cells of mutations that make this typical major intrinsic protein more similar to Fps1p. We observed that mutations made in Fps1p to "restore" the signature NPA motifs did not substantially affect channel function. In contrast, when GlpF was mutated to resemble Fps1p, all mutants had reduced activity compared with wild type. We rationalized these data by constructing models of one GlpF mutant and of the transmembrane core of Fps1p. Our model predicts that the pore of Fps1p is more flexible than that of GlpF. We discuss the fact that this may accommodate the divergent NPA motifs of Fps1p and that the different pore structures of Fps1p and GlpF may reflect the physiological roles of the two glycerol facilitators.

A novel role of plasma membrane calcium atpase 4 as a negative-regulator of VEGF-induced angiogenesis

Current anti-angiogenic treatments involve the attenuation of signalling via the pro-angiogenic vascular endothelial growth factor/receptor (VEGF/VEGFR) axis. Stimulation of angiogenesis by VEGF requires the activation of the calcineurin/nuclear factor of activated T-cells (NFAT) signal transduction pathway which is inhibited by Plasma Membrane Calcium ATPase 4 (PMCA4), an endogenous calcium extrusion pump. However, PMCA4s role in calcineurin/NFAT-dependent angiogenesis is unknown. Using “gain of function” studies, we show here that adenoviral overexpression of PMCA4 in human umbilical vein endothelial cells (HUVEC) inhibited NFAT activity, decreased the expression of NFAT-dependent pro-angiogenic proteins (regulator of calcineurin 1.4 (RCAN1.4) and cyclooxygenase-2) and diminished in vitro cell migration and tube formation in response to VEGF-stimulation. Furthermore, in vivo blood vessel formation was attenuated in a matrigel plug assay by ectopic expression of PMCA4. Conversely, “loss of function” experiments by si-RNA-mediated knockdown of PMCA4 in HUVEC or isolation of mouse lung endothelial cells from PMCA4−/− mice showed increased VEGF-induced NFAT activity, RCAN1.4 expression, in vitro endothelial cell migration, tube formation and in vivo blood vessel formation. Additionally, in an in vivo pathological angiogenesis model of limb ischemia, the reperfusion of the ischemic limb of PMCA4−/− mice was augmented compared to wild-type. Disruption of the interaction between endogenous PMCA4 and calcineurin by adenoviral overexpression of the region of PMCA4 that interacts with calcineurin (residues 428–651) increased NFAT activity, RCAN1.4 protein expression and in vitro tube formation. These results identify PMCA4 as an inhibitor of VEGF-induced angiogenesis, highlighting its potential as a new therapeutic target for anti-angiogenic treatments.