Use of synthetic CaV2.2 Ca2+ channel peptides to study presynaptic function

Small, synthetic peptides based on specific regions of voltage-gated Ca2+ channels (VGCCs) have been widely used to study Ca2+ channel function and have been instrumental in confirming the contribution of specific amino acid sequences to interactions with putative binding partners. In particular, peptides based on the Ca2+ channel Alpha Interaction Domain (AID) on the intracellular region connecting domains I and II (the I-II loop) and the SYNaptic PRotein INTerction (synprint) site on the II-III loop have been widely used. Emerging evidence suggests that such peptides may themselves possess inherent functionality, a property that may be exploitable for future drug design. Here, we review our recent work using synthetic Ca2+ channel peptides based on sequences within the CaV2.2 amino terminal and I-II loop, originally identified as molecular determinates for G protein modulation, and their effects on VGCC function. These CaV2.2 peptides act as inhibitory modules to decrease Ca2+ influx with direct effects on VGCC gating, ultimately leading to a reduction of synaptic transmission. CaV2.2 peptides also attenuate G protein modulation of VGCCs. Amino acid substitutions generate CaV2.2 peptides with increased or decreased inhibitory effects suggesting that synthetic peptides can be used to further probe VGCC function and, potentially, form the basis for novel therapeutic development.

Synaptic vesicle glycoprotein 2A modulates vesicular release and calcium channel function at peripheral sympathetic synapses

Synaptic vesicle glycoprotein (SV)2A is a transmembrane protein found in secretory vesicles and is critical for Ca2+-dependent exocytosis in central neurons, although its mechanism of action remains uncertain. Previous studies have proposed, variously, a role of SV2 in the maintenance and formation of the readily releasable pool (RRP) or in the regulation of Ca2+ responsiveness of primed vesicles. Such previous studies have typically used genetic approaches to ablate SV2 levels; here, we used a strategy involving small interference RNA (siRNA) injection to knockdown solely presynaptic SV2A levels in rat superior cervical ganglion (SCG) neuron synapses. Moreover, we investigated the effects of SV2A knockdown on voltage-dependent Ca2+ channel (VDCC) function in SCG neurons. Thus, we extended the studies of SV2A mechanisms by investigating the effects on vesicular transmitter release and VDCC function in peripheral sympathetic neurons. We first demonstrated an siRNA-mediated SV2A knockdown. We showed that this SV2A knockdown markedly affected presynaptic function, causing an attenuated RRP size, increased paired-pulse depression and delayed RRP recovery after stimulus-dependent depletion. We further demonstrated that the SV2A–siRNA-mediated effects on vesicular release were accompanied by a reduction in VDCC current density in isolated SCG neurons. Together, our data showed that SV2A is required for correct transmitter release at sympathetic neurons. Mechanistically, we demonstrated that presynaptic SV2A: (i) acted to direct normal synaptic transmission by maintaining RRP size, (ii) had a facilitatory role in recovery from synaptic depression, and that (iii) SV2A deficits were associated with aberrant Ca2+ current density, which may contribute to the secretory phenotype in sympathetic peripheral neurons.

Developmental changes in presynaptic muscarinic modulation of excitatory and inhibitory neurotransmission in rat piriform cortex in vitro: relevance to epileptiform bursting susceptibility

Suppression of depolarizing postsynaptic potentials and isolated GABA-A receptor-mediated fast inhibitory postsynaptic potentials by the muscarinic acetylcholine receptor agonist, oxotremorine-M (10 microM), was investigated in adult and immature (P14-P30) rat piriform cortical (PC) slices using intracellular recording. Depolarizing postsynaptic potentials evoked by layers II-III stimulation underwent concentration-dependent inhibition in oxotremorine-M that was most likely presynaptic and M2 muscarinic acetylcholine receptor-mediated in immature, but M1-mediated in adult (P40-P80) slices; percentage inhibition was smaller in immature than in adult piriform cortex. In contrast, compared with adults, layer Ia-evoked depolarizing postsynaptic potentials in immature piriform cortex slices in oxotremorine-M, showed a prolonged multiphasic depolarization with superimposed fast transients and spikes, and an increased 'all-or-nothing' character. Isolated N-methyl-d-aspartate receptor-mediated layer Ia depolarizing postsynaptic potentials (although significantly larger in immature slices) were however, unaffected by oxotremorine-M, but blocked by dl-2-amino-5-phosphonovaleric acid. Fast inhibitory postsynaptic potentials evoked by layer Ib or layers II-III-fiber stimulation in immature slices were significantly smaller than in adults, despite similar estimated mean reversal potentials ( approximately -69 and -70 mV respectively). In oxotremorine-M, only layer Ib-fast inhibitory postsynaptic potentials were suppressed; suppression was again most likely presynaptic M2-mediated in immature slices, but M1-mediated in adults. The degree of fast inhibitory postsynaptic potential suppression was however, greater in immature than in adult piriform cortex. Our results demonstrate some important physiological and pharmacological differences between excitatory and inhibitory synaptic systems in adult and immature piriform cortex that could contribute toward the increased susceptibility of this region to muscarinic agonist-induced epileptiform activity in immature brain slices.

G-protein-coupled-receptor-mediated presynaptic inhibition in the cerebellum

Throughout the central nervous system a dominant form of inhibition of neurotransmitter release from presynaptic terminals is mediated by G-protein-coupled receptors (GPCRs). Neurotransmitter release is typically induced by action potentials (APs), but can also occur spontaneously. Presynaptic inhibition by GPCRs has been associated with modulation of voltage-dependent ion channels. However, electrophysiological recordings of spontaneous, AP-independent (so-called ‘miniature’) postsynaptic events reveal an additional, important form of GPCR-mediated presynaptic inhibition, distinct from effects on ionic conductances and consistent with a direct action on the vesicle release machinery. Recent studies suggest that such miniature events might be of physiological relevance not only in signalling but also in development. In the cerebellum, neurotransmitter release onto Purkinje cells occurs by AP-dependent and AP-independent pathways. Here, I focus on inhibitory synapses between interneurons and Purkinje cells, which are subject to strong, identifiable regulation by endogenous GPCR agonists, to consider mechanisms of GPCR-mediated presynaptic inhibition.

Calcium carbonate solubilization through H-proton release from some legumes grown in calcareous saline-sodic soils

Increasing levels of CO2 and H+ proton in the rhizosphere from some legumes may play an important role in calcite dissolution of calcareous salt affected soils. Soils planted with white and brown varieties of cowpea (Vigna unguiculata L.) and hyacinth bean (Dolichos lablab L.) relying on either fertilizer N (KNO3) or N-fixation were compared against soils to which gypsum was applied and a control without plants and gypsum application to study the possibility of Ca2+ release from calcite and Na+ leaching. As compared to plants relying on inorganic N, leachates from all pore volumes (0·5, 1·0, 1·5, 2·0 pore volume) in lysimeters planted with N-fixing hyacinth bean contained significantly higher concentrations of HCO with lower concentrations from lysimeters planted with white cowpea relying on N-fixation. However, the lowest concentrations of HCO were recorded in the gypsum and control treatments. In initial leaching, lysimeters planted with N-fixing plants maintained similar leachate Ca2+ and Na+ concentrations compared to gypsum amended soils. However, gypsum amended soils were found to have a prolonged positive effect on Na+ removal. It might be concluded that some legumes that are known to fix N in calcareous salt affected soils may be an alternative ameliorant to the extremely expensive gypsum through calcite solubilization and a consequent release of Ca2+.

Tumour necrosis factor alpha induces rapid reduction in AMPA receptor-mediated calcium entry in motor neurones by increasing cell surface expression of the GluR2 subunit: relevance to neurodegeneration

The AMPA receptor (AMPAR) subunit GluR2, which regulates excitotoxicity and the inflammatory cytokine tumour necrosis factor alpha (TNF alpha) have both been implicated in motor neurone vulnerability in Amyotrophic Lateral Sclerosis/Motor Neurone Disease. TNF alpha has been reported to increase cell surface expression of AMPAR subunits to increase synaptic strength and enhance excitotoxicity, but whether this mechanism occurs in motor neurones is unknown. We used primary cultures of mouse motor neurones and cortical neurones to examine the interaction between TNF alpha receptor activation, GluR2 availability, AMPAR-mediated calcium entry and susceptibility to excitotoxicity. Short exposure to a physiologically relevant concentration of TNFalpha (10 ng/ml, 15 min) caused a marked redistribution of both GluR1 and GluR2 to the cell surface as determined by cell surface biotinylation and immunofluorescence. Using Fura-2 AM microfluorimetry we showed that exposure to TNFalpha caused a rapid reduction in the peak amplitude of AMPA-mediated calcium entry in a PI3-kinase and p38 kinase-dependent manner, consistent with increased insertion of GluR2-containing AMPAR into the plasma membrane. This resulted in a protection of motor neurones against kainate-induced cell death. Our data therefore, suggests that TNF alpha acts primarily as a physiological regulator of synaptic activity in motor neurones rather than a pathological drive in ALS

Comparison of ammonium lactate, sodium bicarbonate and double calcium lactate methods for extraction of phosphorus from wetland peat soils

Procedures for routine analysis of soil phosphorus (P) have been used for assessment of P status, distribution and P losses from cultivated mineral soils. No similar studies have been carried out on wetland peat soils. The objective was to compare extraction efficiency of ammonium lactate (PAL), sodium bicarbonate (P-Olsen), and double calcium lactate (P-DCaL) and P distribution in the soil profile of wetland peat soils. For this purpose, 34 samples of the 0-30, 30-60 and 60-90 cm layers were collected from peat soils in Germany, Israel, Poland, Slovenia, Sweden and the United Kingdom and analysed for P. Mean soil pH (CaCl2, 0.01 M) was 5.84, 5.51 and 5.47 in the 0-30, 30-60 and 60-90 cm layers, respectively. The P-DCaL was consistently about half the magnitude of either P-AL or P-Olsen. The efficiency of P extraction increased in the order P-DCaL < P-AL &LE; P-Olsen, with corresponding means (mg kg(-1)) for all soils (34 samples) of 15.32, 33.49 and 34.27 in 0-30 cm; 8.87, 17.30 and 21.46 in 30-60 cm; and 5.69, 14.00 and 21.40 in 60-90 cm. The means decreased with depth. When examining soils for each country separately, P-Olsen was relatively evenly distributed in the German, UK and Slovenian soils. P-Olsen was linearly correlated (r = 0.594, P = 0.0002) with pH, whereas the three P tests (except P-Olsen vs P-DCaL) significantly correlated with each other (P = 0.017850.0001). The strongest correlation (r = 0.617, P = 0.0001) was recorded for P-AL vs P-DCaL) and the two methods were inter-convertible using a regression equation: P-AL = -22.593 + 5.353 pH + 1.423 P-DCaL, R-2 = 0.550.

Uneven distribution of nutrients in the root zone affects the incidence of blossom end rot and concentration of calcium and potassium in fruits of tomato

Tomato plants ( Lycopersicon esculentum Mill. var. DRK) were grown hydroponically to determine the effect of an uneven distribution of nutrients in the root zone on blossomend rot (BER) and Ca and K concentrations in the fruits. The plants were grown in rockwool with their root system divided into two portions. Each portion was irrigated with nutrient solutions with either the same or the different electrical conductivity (EC) in the range 0 to 6 dS m(-1). Solutions with high EC supplied to both sides of the root system significantly increased the incidence of BER. However, when only water or a solution of low EC was supplied to one portion, BER was reduced by 80%. Fruit yields were significantly higher ( P < 0.01) for plants that received solutions of the uneven EC treatments (6/0 or 4.5/0 EC treatment). Plants supplied with solutions of uneven EC generally had higher leaf and fruit concentrations of Ca but lower concentrations of K than those supplied with solutions of high EC. There was no difference in Ca concentration at the distal end of young fruits of the uneven EC treatment but it was reduced in the high EC treatments. The concentration of K in the mature fruits of the uneven EC treatments was lower than that of the high EC treatments and higher or similar that of the 3/3 or 2.5/2.5 EC treatments ( controls). A clear relationship was found between the incidence of BER and the exudation rate. High rate of xylem exudation was observed in the uneven EC treatments. Reduction of BER in the uneven EC treatments is most likely to be the effect of high exudation rate on Ca status in the young fruits. It was concluded that high EC of solution had positive effects on Ca concentration and incidence of BER provided that nutrient solution with low EC or water is supplied to the one portion of the root system.

The effect of calcium removal from milk on casein micelle stability and structure

The total calcium level of raw skimmed milk was reduced by 10, 19, 29, 40 and 51% using Duolite® ion-exchange resin. The products were examined for concentrations of ionic calcium, sodium and potassium and the pH, ethanol stability, micelle diameter and ζ-potential were also measured. Ionic calcium decreased with removal of calcium and pH increased. Calcium removal resulted in an increase in the ethanol stability from 88% to above 100%. Casein micelle diameter increased as calcium was removed. The ζ-potential of the skimmed bulk milk was -24.4 mV, gradually becoming more negative with calcium removal to -30.6 mV after 51% calcium removal. The milk became more translucent as calcium was removed. To investigate the reversibility of this process, calcium chloride was added back to the depleted samples to restore their original total calcium content. At 51% removal, restoration of the total calcium level resulted in formation of clots. At levels of 10 and 19% calcium removal, the ethanol stability remained above 100%, but at higher levels of calcium removal the alcohol stability was adversely affected when the calcium was added back. Adding back calcium resulted in partial restoration of the original casein micelle diameter.

Effects of different calcium salts on properties of milk related to heat stability

Insoluble calcium salts were added to milk to increase total calcium by 30 mM, without changing properties influencing heat stability, such as pH and ionic calcium. There were no major signs of instability associated with coagulation, sediment formation or fouling when subjected to ultra high temperature (UHT) and in-container sterilisation. The buffering capacity was also unaltered. On the other hand, addition of soluble calcium salts reduced pH, increased ionic calcium and caused coagulation to occur. Calcium chloride showed the largest destabilising effect, followed by calcium lactate and calcium gluconate. Milk became unstable to UHT processing at lower calcium additions compared to in-container sterilisation.

Measurement of ionic calcium, pH and soluble divalent cations in milk at high temperature

Dialysis and ultrafiltration were investigated as methods for measuring pH and ionic calcium and partitioning of divalent cations of milk at high temperatures. It was found that ionic calcium, pH, and total soluble divalent cations decreased as temperature increased between 20 and 80°C in both dialysates and ultrafiltration permeates. Between 90 and 110°C, ionic calcium and pH in dialysates continued to decrease as temperature increased, and the relationship between ionic calcium and temperature was linear. The permeabilities of hydrogen and calcium ions through the dialysis tubing were not changed after the tubing was sterilized for 1h at 120°C. There were no significant differences in pH and ionic calcium between dialysates from raw milk and those from a range of heat-treated milks. The effects of calcium chloride addition on pH and ionic calcium were measured in milk at 20°C and in dialysates collected at 110°C. Heat coagulation at 110°C occurred with addition of calcium chloride at 5.4mM, where pH and ionic calcium of the dialysate were 6.00 and 0.43mM, respectively. Corresponding values at 20°C were pH 6.66 and 2.10mM.

Ionic calcium and pH as predictors of stability of milk to UHT processing

This paper describes the use of pH and calcium ion electrodes for investigating factors affecting the heat stability of UHT milk with added calcium chloride. Calcium chloride was added to raw milk to manipulate ionic calcium and pH to within the range that may be typically encountered in raw milk of different compositions and microbial quality. Addition of only 5 mM calcium chloride was sufficient to induce considerable changes in pH, ionic calcium and ethanol stability and alter its stability to UHT treatment. There was a strong relationship between pH decrease and increase in ionic calcium when pH was reduced, whether by addition of calcium chloride or by acidification. Calcium chloride addition was found to increase sediment formation in UHT treated milk. However, sediment could be reduced by addition of stabilizers. Those most effective were ones which decreased ionic calcium and increased pH, such as trisodium citrate and disodium hydrogen phosphate. Sediment formation following UHT treatment was only slight for milk samples whose ethanol stability was greater than 80%.

Effect of replacing calcium salts of palm oil distillate with rapeseed oil, milled or whole rapeseeds on milk fatty-acid composition in cows fed maize silage-based diets

Inclusion of rapeseed feeds in dairy cow diets has the potential to reduce milk fat saturated fatty acid (SFA) and increase cis-monounsaturated fatty acid (cis-MUFA) content but effectiveness may depend on the form in which the rapeseed is presented. Four mid-lactation Holstein dairy cows were allocated to four maize silage-based dietary treatments according to a 4 x 4 Latin Square design, with 28-day experimental periods. Treatments consisted of a control diet (C containing 49 g/kg dry matter (DM) of calcium salts of palm oil distillate (CPO), or 49 g/kg DM of oil supplied as whole rapeseeds (WR), rapeseeds milled with wheat (MR) or rapeseed oil (RO). Replacing CPO with rapeseed feeds had no effect (P > 0.05) on milk fat and protein content, while milk yields were higher (P < 0.05) for RO and MR compared with WR (37.1, 38.1 and 34.3 kg/day, respectively). Substituting CPO with RO or MR reduced (P < 0.05) milk fat total SFA content (69.6, 55.6, 71.7 and 61.5 g/100g fatty acids for C, RO, WR and MR, respectively) and enhanced (P < 0.05) milk cis-9 18:1 MUFA concentrations (corresponding values 18.6, 24.3, 17.0 and 23.0 g/100g fatty acids) compared with C and WR. Treatments RO and MR also increased (P < 0.05) milk trans-MUFA content (4.4, 6.8, 10.5 g/100g fatty acids, C MR and RO, respectively). A lack of significant changes in milk fat composition when replacing CPO with WR suggests limited bioavailability of fatty acids in intact rapeseeds. In conclusion, replacing a commercial palm oil-based fat supplement in the diet with milled rapeseeds or rapeseed oil represented an effective strategy to alter milk fatty acid composition with the potential to improve human health. Inclusion of processed rapeseeds offered a good compromise for reducing milk SFA and increasing cis-MUFA, whilst minimising milk trans-MUFA and negative effects on animal performance.