Effects of Pb<Superscript>2+</Superscript> on the active oxygen-scavenging enzyme activities and ultrastructure in <Emphasis Type="Italic">Potamogeton crispus</Emphasis> leaves

The effects of various concentrations of Pb²⁺ on the antioxidant enzyme activities and the ultrastructure in Potamogeton crispus leaves were studied. Peroxidase (POD) activity and malondialdehyde (MDA) content peaks were observed with an increase in Pb²⁺ concentration, whereas superoxide dismutase (SOD) and catalase (CAT) activities decreased firstly and then rose. Meantime, the chlorophyll content declined with increasing Pb²⁺ concentration. Simultaneously, high concentrations of Pb²⁺ aggravated ultrastructural damage to the leaf cells including swelling of chloroplasts, disruption and disappearance of chloroplast envelopes; swelling of mitochondrial cristae, deformation and vacuolation of mitochondria; condensation of chromatin, dispersion of nucleoli, and disruption of nuclear membrane. Changes in antioxidant enzyme activities and damage to fine structure are the results of lead-induced ROS accumulation. The estimated lethal concentration to P. crispus ranged from 10 to 15 mg/l lead. Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 3, pp. 469–474. The text was submitted by the authors in English.     

Signaling Pathways in the Biphasic Effect of ANG II on Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> Exchanger in T84 Cells

The effect of ANG II on pH_i, [Ca²⁺]_i and cell volume was investigated in T84 cells, a cell line originated from colon epithelium, using the probes BCECF-AM, Fluo 4-AM and acridine orange, respectively. The recovery rate of pH_i via the Na⁺/H⁺ exchanger was examined in the first 2 min following the acidification of pH_i with a NH₄Cl pulse. In the control situation, the pH_i recovery rate was 0.118 ± 0.001 (n = 52) pH units/min and ANG II (10⁻¹² M or 10⁻⁹ M) increased this value (by 106% or 32%, respectively) but ANG II (10⁻⁷ M) decreased it to 47%. The control [Ca²⁺]_i was 99 ± 4 (n = 45) nM and ANG II increased this value in a dose-dependent manner. The ANG II effects on cell volume were minor and late and should not interfere in the measurements of pH_i recovery and [Ca²⁺]_i. To document the signaling pathways in the hormonal effects we used: Staurosporine (a PKC inhibitor), W13 (a calcium-dependent calmodulin antagonist), H89 (a PKA inhibitor) or Econazole (an inhibitor of cytochrome P450 epoxygenase). Our results indicate that the biphasic effect of ANG II on Na⁺/H⁺ exchanger is a cAMP-independent mechanism and is the result of: 1) stimulation of the exchanger by PKC signaling pathway activation (at 10⁻¹² – 10⁻⁷ M ANG II) and by increases of [Ca²⁺]_i in the lower range (at 10⁻¹² M ANG II) and 2) inhibition of the exchanger at high [Ca²⁺]_i levels (at 10⁻⁹ – 10⁻⁷ M ANG II) through cytochrome P450 epoxygenase-dependent metabolites of the arachidonic acid signaling pathway.     

Chronic Nicotine Alters Nicotinic Receptor-induced Presynaptic Ca<Superscript>2+</Superscript> Responses in Isolated Nerve Terminals

Brain nicotinic receptors display pronounced permeability for Ca²⁺ and localize to presynaptic nerve terminals, in addition to postsynaptic sites. Chronic exposure to nicotine has been shown to alter brain nicotinic receptor expression, but the functional consequences for presynaptic Ca²⁺ have not been directly examined. Here, we used confocal imaging to assess Ca²⁺ responses in individual nerve terminals from cortices of mice treated up to 14 days with nicotine as compared to vehicle-treated controls. Chronic nicotine treatment led to substantially enhanced amplitudes of presynaptic Ca²⁺ responses to acute application of nicotine at concentrations of 50 nM (2-fold) and 500 nM (1.7-fold), but not 50 μM. In addition, increased expression of high-affinity nicotinic receptors on isolated terminals was observed following chronic treatment, as determined immunocytochemically and pharmacologically. These findings suggest that chronic exposure to nicotine may lead to enhanced sensitivity to nicotine at select presynaptic sites in brain via up-regulation of high-affinity nicotinic receptors.     

Effects of Pb<Superscript>2+</Superscript> ions on Na<Superscript>+</Superscript> transport in the isolated skin of the toad <Emphasis Type="Italic">Pleurodema thaul</Emphasis>

The effects induced by lead ions on the short-circuit current (SCC) and on the potential difference (V) of the toad Pleurodema thaul skin were investigated. Pb²⁺ applied to the outer (mucosal) surface increased SCC and V and when applied to the inner (serosal) surface decreased both parameters. The stimulatory effect, but not the inhibitory action, was reversible after washout of the metal ion. The amiloride test showed that the increase was due principally to stimulation of the driving potential for Na⁺ (V-E_Na+) and that inhibition was accompanied by a reduction in the V-E_Na+ and also by a significant decrease in skin resistance indicating possible disruption of membrane and/or cell integrity. The effect of noradrenaline was increased by outer and decreased by inner administration of Pb²⁺. The results suggest that mucosal Pb²⁺ activates toad skin ion transport by stimulating the V-E_Na+ and that serosal Pb²⁺, with easier access to membrane and cellular constituents, inactivates this mechanism, revealing greater toxicity when applied to the inner surface of the skin. Abbreviations: SCC – short-circuit current; V – potential difference; V-E_Na+– driving potential for Na⁺; ENaC – epithelial sodium channel; R_Na+– active sodium resistance; RS – passive or shunt resistance; G_Na– active sodium conductance; GS – passive or shunt conductance; Gmax – total conductance; EC₅₀– half-maximal excitatory concentration; IC₅₀– half maximal inhibitory concentration; NA – noradrenaline.     

Neuromuscular transmission in Ca<Superscript>2+</Superscript>-free extracellular solution

The review brings together the data on neuromuscular transmission upon substitution of different alkaline earth metals for Ca²⁺ ions. It is known that due to the low selectivity of calcium channels and their ability to conduct other divalent cations, a considerable presynaptic current carried by strontium or barium may develop, which under certain conditions may lead to the neuromuscular transmission. The review illustrates how the equimolar substitution of external Ca²⁺ by other polyvalent cations affects the parameters of nonquantum, spontaneous, and induced quantum exocytosis of the neuromediator, as well as endocytosis and the activities of acetylcholinesterase and postsynaptic receptors. The effects of the modulators of synaptic transmission under these conditions are also considered.     

Presynaptic Mechanisms of Lead Neurotoxicity: Effects on Vesicular Release,Vesicle Clustering and Mitochondria Number

Childhood lead (Pb²⁺) intoxication is a global public health problem and accounts for 0.6% of the global burden of disease associated with intellectual disabilities. Despite the recognition that childhood Pb²⁺ intoxication contributes significantly to intellectual disabilities, there is a fundamental lack of knowledge on presynaptic mechanisms by which Pb²⁺ disrupts synaptic function. In this study, using a well-characterized rodent model of developmental Pb²⁺ neurotoxicity, we show that Pb²⁺ exposure markedly inhibits presynaptic vesicular release in hippocampal Schaffer collateral-CA1 synapses in young adult rats. This effect was associated with ultrastructural changes which revealed a reduction in vesicle number in the readily releasable/docked vesicle pool, disperse vesicle clusters in the resting pool, and a reduced number of presynaptic terminals with multiple mitochondria with no change in presynaptic calcium influx. These studies provide fundamental knowledge on mechanisms by which Pb²⁺ produces profound inhibition of presynaptic vesicular release that contribute to deficits in synaptic plasticity and intellectual development.     

Microcalorimetric Study on the Toxic Effect of Pb<Superscript>2+</Superscript> to <Emphasis Type="Italic">Tetrahymena</Emphasis>

The toxic effect of Pb²⁺ has been studied in eukaryotic cells by using Tetrahymena as a target. The maximum power (P _m) and the growth rate constant (k) were determined, which showed that values of P _m and k were linked to the concentration (C) of Pb²⁺. The addition of Pb²⁺ caused a decrease of the maximum heat production and growth rate constant, indicating that Tetrahymena growth was inhibited in the presence of Pb²⁺, and Pb²⁺ took part in the metabolism of cells. From micrographs, morphological changes of Tetrahymena were observed with addition of Pb²⁺, indicating that the toxic effect of Pb²⁺ derived from destroying the membrane of surface of Tetrahymena. According to the thermogenic curves and photos of Tetrahymena under different conditions, it is clear that metabolic mechanism of Halobacterium halobium R1 growth has been changed with the addition of Pb²⁺.     

Significance of the degree of synaptic Zn<Superscript>2+</Superscript> signaling in cognition

Zinc is a trace nutrient for the brain and a signal factor to serve for brain function. A portion of zinc is released from glutamatergic (zincergic) neuron terminals in the brain. Synaptic Zn²⁺ signaling is involved in synaptic plasticity such as long-term potentiaion (LTP), which is a cellular mechanism of memory. The block and/or loss of synaptic Zn²⁺ signaling in the hippocampus and amygdala with Zn²⁺ chelators affect cognition, while the role of synaptic Zn²⁺ signal is poorly understood, because zinc-binding proteins are great in number and multi-functional. Chronic zinc deficiency also affects cognition and cognitive decline induced by zinc deficiency might be associated with the increase in plasma glucocorticoid rather than the decrease in synaptic Zn²⁺ signaling. On the other hand, excess glutamatergic (zincergic) neuron activity induces excess influx of extracellular Zn²⁺ into hippocampal neurons, followed by cognitive decline. Intracellular Zn²⁺ dynamics, which is linked to presynaptic glutamate release, is critical for LTP and cognitive performance. This paper deals with insight into cognition from zinc as a nutrient and signal factor.     

Calcium-induced modulation of synaptic transmission

Calcium (Ca²⁺) is a second messenger regulating a wide variety of intracellular processes. Using GABA-and glycinergic synapses as examples, this review analyzes two functions of this unique ion: postsynaptic Ca²⁺-dependent modulation of receptor-operated channels and Ca²⁺-induced retrograde regulation of neurotransmitter release from the presynaptic terminals. Phosphorylation, rapid Ca²⁺-induced modulation via intermediate Ca²⁺-binding proteins, and changes in the number of functional receptors represent the main pathways of short-and long-term plasticity of postsynaptic receptor-operated channel machinery. Retrograde signaling is an example of synaptic modulation triggered by stimulation of postsynaptic cells and mediated via regulation of presynaptic neurotransmitter release. This mechanism provides postsynaptic neurons with efficient tools to control the presynaptic afferents in an activity-dependent mode. Elevation of intracellular Ca²⁺ in a postsynaptic neuron triggers the synthesis of endocannabinoids (derivatives of arachidonic acid). Their retrograde diffusion through the synaptic cleft and consequent activation of presynaptic G-protein coupled to CB1 receptors inhibits the release of neurotransmitter. These mechanisms of double modulation, which include control over the function of postsynaptic ion channels and retrograde suppression of the release machinery, play an important role in Ca²⁺-dependent control of the main excitatory and inhibitory synaptic pathways in the mammalian nervous system.     

Resonance Scattering Spectral Detection of Trace Pb<Superscript>2+</Superscript> Using Aptamer-Modified AuPd Nanoalloy as Probe

The resonance scattering spectral probe for Pb²⁺ was obtained using aptamer-modified AuPd Nanoalloy. In the pH 7.0 Na₂HPO₄–NaH₂PO₄ buffer solution, the aptamer interacted with AuPd nanoalloy particles to form stable aptamer-AuPd nanoalloy probe for Pb²⁺ that is stable in high concentration of salt. The probe combined with Pb²⁺ ions to form a G-quadruplex and to release AuPd nanoalloy particles that aggregate to form big particles which led the resonance scattering (RS) intensity enhancing. The reaction solution was filtered by 0.15 μm membrane to obtain the filtration containing aptamer-AuPd nanoalloy probe that has strong catalytic effect on the electrodeless nickel particle plating reaction between Ni(II) and PO₂³⁻ that exhibited a strong RS peak at 508 nm. The RS intensity at 508 nm decreased when the Pb²⁺ concentration increased. The decreased intensity (ΔI _508nm) is linear to the concentration of 0.08–42 nM Pb²⁺, with regress equation of DI_508nm = 16.3 c + 1.5 \Delta {I_{{5}0{\rm{8nm}}}} = {16}.{3}\,c + {1}.{5} , correlation coefficient of 0.9965, and detection limit of 0.04 nM Pb²⁺. The RS assay was applied to the analysis of Pb²⁺ in wastewater, with satisfactory results.     

Ca<Superscript><Emphasis Type="Italic">2+</Emphasis></Superscript>-Induced Cl<Superscript>−</Superscript> Efflux at Rat Distal Colonic Epithelium

With the aid of the halide-sensitive dye 6-methoxy-N-ethylquinolinium iodide (MEQ), changes in intracellular Cl^- concentration were measured to characterize the role of Ca²⁺-dependent Cl^- channels at the rat distal colon. In order to avoid indirect effects of secretagogues mediated by changes in the driving force for Cl^- exit (i.e., mediated by opening of Ca²⁺-dependent K⁺ channels), all experiments were performed under depolarized conditions, i.e., in the presence of high extracellular K⁺ concentrations. The Ca²⁺-dependent secretagogue carbachol induced a stilbene-sensitive Cl^- efflux, which was mimicked by the Ca²⁺ ionophore ionomycin. Surprisingly, the activation of Ca²⁺-dependent Cl^- efflux was resistant against blockers of classical Ca²⁺ signaling pathways such as phospholipase C, protein kinase C and calmodulin. Hence, alternative pathways must be involved in the signaling cascade. One possible signaling molecule seems to be nitric oxide (NO) as the NO donor sodium nitroprusside could induce Cl^- efflux. Vice versa, the NO synthase inhibitor N-ω-monomethyl-arginine (l-NMMA) reduced the carbachol-induced Cl^- efflux. This indicates that NO may be involved in part of the signaling cascade. In order to test the ability of the epithelium to produce NO, the expression of different isoforms of NO synthase was verified by immunohistochemistry. In addition, the cytoskeleton seems to play a role in the activation of Ca²⁺-dependent Cl^- channels. Inhibitors of microtubule association such as nocodazole and colchicine as well as jasplakinolide, a drug that enhances actin polymerization, inhibited the carbachol-induced Cl^- efflux. Consequently, the activation of apical Cl^- channels by muscarinic receptor stimulation differs in signal transduction from the classical phospholipase C/protein kinase C way.     

Adhesion forces in the self-recognition of oligosaccharide epitopes of the proteoglycan aggregation factor of the marine sponge <Emphasis Type="Italic">Microciona prolifera</Emphasis>

Cell aggregation in the marine sponge Microciona prolifera is mediated by a multimillion molecular-mass aggregation factor, termed MAF. Earlier investigations revealed that the cell aggregation activity of MAF depends on two functional domains: (i) a Ca²⁺-independent cell-binding domain and (ii) a Ca²⁺-dependent proteoglycan self-interaction domain. Structural analysis of involved carbohydrate fragments of the proteoglycan in the self-association established a sulfated disaccharide β-d-GlcpNAc3S-(1→3)-α-l-Fucp and a pyruvated trisaccharide β-d-Galp4,6(R)Pyr-(1→4)-β-d-GlcpNAc-(1→3)-α-l-Fucp. Recent UV, SPR, and TEM studies, using BSA conjugates and gold nanoparticles of the synthetic sulfated disaccharide, clearly demonstrated self-recognition on the disaccharide level in the presence of Ca²⁺-ions. To determine binding forces of the carbohydrate–carbohydrate interactions for both synthetic MAF oligosaccharides, atomic force microscopy (AFM) studies were carried out. It turned out that, in the presence of Ca²⁺-ions, the force required to separate the tip and sample coated with a self-assembling monolayer of thiol-spacer-containing β-d-GlcpNAc-(1→3)-α-l-Fucp-(1→O)(CH₂)₃S(CH₂)₆S- was found to be quantized in integer multiples of 30 ± 6 pN. No binding was observed between the two monolayers in the absence of Ca²⁺-ions. Cd²⁺-ions could partially induce the self-interaction. In contrast, similar AFM experiments with thiol-spacer-containing β-d-Galp4,6(R)Pyr-(1→4)-β-d-GlcpNAc-(1→3)-α-l-Fucp-(1→O)(CH₂)₃S(CH₂)₆S- did not show a binding in the presence of Ca²⁺-ions. Also TEM experiments of gold nanoparticles coated with the pyruvated trisaccharide could not make visible aggregation in the presence of Ca²⁺-ions. It is suggested that the self-interaction between the sulfated disaccharide fragments is stronger than that between the pyruvated trisaccharide.     

Biosorption of Pb<Superscript>2+</Superscript> and Zn<Superscript>2+</Superscript> by Non-Living Biomass of <Emphasis Type="Italic">Spirulina</Emphasis> sp.

Removal of heavy metals (Pb²⁺, Zn²⁺) from aqueous solution by dried biomass of Spirulina sp. was investigated. Spirulina rapidly adsorbed appreciable amount of lead and zinc from the aqueous solutions within 15 min of initial contact with the metal solution and exhibited high sequestration of lead and zinc at low equilibrium concentrations. The specific adsorption of both Pb²⁺ and Zn²⁺ increased at low concentration and decreased when biomass concentration exceeded 0.1 g l⁻¹. The binding of lead followed Freundlich model of kinetics where as zinc supported Langmuir isotherm for adsorption with their r ² values of 0.9659 and 0.8723 respectively. The adsorption was strongly pH dependent as the maximum lead biosorption occurred at pH 4 and 10 whereas Zn²⁺ adsorption was at pH 8 and 10.     

Gβγ SNARE Interactions and Their Behavioral Effects

Presynaptic terminals possess interlocking molecular mechanisms that control exocytosis. An example of such complexity is the modulation of release by presynaptic G Protein Coupled Receptors (GPCRs). GPCR ubiquity at synapses—GPCRs are present at every studied presynaptic terminal—underlies their critical importance in synaptic function. GPCRs mediate presynaptic modulation by mechanisms including via classical Gα effectors, but membrane-delimited actions of Gβγ can also alter probability of release by altering presynaptic ionic conductances. This directly or indirectly modifies action potential-evoked presynaptic Ca²⁺ entry. In addition, Gβγ can interact directly with SNARE complexes responsible for synaptic vesicle fusion to reduce peak cleft neurotransmitter concentrations during evoked release. The interaction of Gβγ with SNARE is displaced via competitive interaction with C2AB-domain containing calcium sensors such as synaptotagmin I in a Ca²⁺-sensitive manner, restoring exocytosis. Synaptic modulation of this form allows selective inhibition of postsynaptic receptor-mediated responses, and this, in combination with Ca²⁺ sensitivity of Gβγ effects on SNARE complexes allows for specific behavioral outcomes. One such outcome mediated by 5-HT receptors in the spinal cord seen in all vertebrates shows remarkable synergy between presynaptic effects of Gβγ and postsynaptic 5-HT-mediated changes in activation of Ca²⁺-dependent K⁺ channels. While acting through entirely separate cellular compartments and signal transduction pathways, these effects converge on the same effect on locomotion and other critical functions of the central nervous system.

     

Novel type of ion channel activated by Pb2+, Cd2+, and Al3+ in cultured mouse neuroblastoma cells

Summary Superfusion with Pb²⁺ induces a slow, noninactivating and reversible inward current in voltage-clamped N1E-115 neuroblastoma cells. The amplitude of this inward current increases in the range of 1–200 m Pb²⁺. Single-channel patch-clamp experiments have revealed that this inward current is mediated by discrete ion channels. Reversal potentials from linearI–V relationships are close to 0 mV for whole-cell and single-channel currents and the single-channel conductance amounts to 24 pS. The Pb²⁺-induced membrane current is not mediated by various known types of ion channels, since it is not blocked by external tetrodotoxin, tetraethylammonium,d-tubocurarine, atropine, ICS 205-930 and by internal EGTA. In Na⁺-free solutions superfusion with Pb²⁺ neither evokes a whole-cell inward current, nor single-channel openings. At –80 mV the open-time distribution of the single channels activated by 1m Pb²⁺ is dual exponential with time constants of 17 and 194 msec. When the Pb²⁺ concentration is increased from 1 to 20 m these time constants decrease to 2 and 13 msec, but the amplitude of single-channel currents remains –1.9 nA. Cd²⁺ and Al³⁺ induce inward currents and single-channel openings similar to Pb²⁺. Time constants fitted to the open-time distribution of single channels are 14 and 135 msec in the presence of 1 m Cd²⁺ and 15 and 99 msec in the presence of 50 m Al³⁺. Conversely, Cu²⁺ induces an irreversible inward current in neuroblastoma cells. Single-channel openings are undetected in the presence of Cu²⁺ and in Na⁺-free solutions Cu²⁺ is still able to induce an inward current. It is concluded that Pb²⁺, Cd²⁺ and possibly Al³⁺ activate a novel type of metal ionactivated (MIA) channel in N1E-115 cells.     

Effects of Levetiracetam,Carbamazepine, Phenytoin,Valproate, Lamotrigine,Oxcarbazepine, Topiramate,Vinpocetine and Sertraline on Presynaptic Hippocampal Na<Superscript>+</Superscript> and Ca<Superscript>2+</Superscript> Channels Permeability

Ion channels are targets of various antiepileptic drugs. In cerebral presynaptic nerve endings Na⁺ and Ca²⁺ channels are particularly abundant, as they control neurotransmitter release, including the release of glutamate (Glu), the most concentrated excitatory amino acid neurotransmitter in the brain. Several pre-synaptic channels are implicated in the mechanism of action of the pro-convulsive agent, 4-aminopyridine (4-AP). In the present study the effects of levetiracetam and other established and newer (vinpocetine) anti-epileptic drugs, as well as of the anti-depressant, sertraline on the increase in Ca²⁺ induced by 4-AP in hippocampal isolated nerve endings were investigated. Also the effects of some of the anti-seizure drugs on the selective increase in Ca²⁺ induced by high K⁺, or on the selective increase in Na⁺ induced by veratridine were tested. Sertraline and vinpocetine effectively inhibited the rise in Ca²⁺ induced by 4-AP, which was dependent on the out-in Na⁺ gradient and tetrodotoxin sensitive. Carbamazepine, phenytoin, lamotrigine and oxcarbazepine inhibited the rise in Ca²⁺ induced by 4-AP too, but at higher concentrations than sertraline and vinpocetine, whereas levetiracetam, valproic acid and topiramate did not. The three latter antiepileptic drugs also failed in modifying other responses mediated by the activation of brain presynaptic Na⁺ or Ca²⁺ channels, including Glu release. This indicates that levetiracetam, valproic acid and topiramate mechanisms of action are unrelated with a decrease in presynaptic Na⁺ or Ca²⁺ channels permeability. It is concluded that depolarized cerebral isolated nerve endings represent a useful tool to unmask potential antiepileptic drugs targeting presynaptic Na⁺ and/or Ca²⁺ channels in the brain; such as vinpocetine or the anti-depressant sertraline, which high effectiveness to control seizures in the animal in vivo has been demonstrated.     

Synaptically Activated Ca<Superscript>2+</Superscript> Release From Internal Stores in CNS Neurons

Synaptically activated postsynaptic [Ca²⁺]_i increases occur through three main pathways: Ca²⁺ entry through voltage-gated Ca²⁺ channels, Ca²⁺ entry through ligand-gated channels, and Ca²⁺ release from internal stores. The first two pathways have been studied intensively; release from stores has been the subject of more recent investigations.Ca²⁺ release from stores in CNS neurons primarily occurs as a result of IP₃ mobilized by activation of metabotropic glutamatergic and/or cholingergic receptors coupled to PLC. Ca²⁺ release is localized near spines in Purkinje cells and occurs as a wave in the primary apical dendrites of pyramidal cells in the hippocampus and cortex. The amplitude of the [Ca²⁺]_i increase can reach several micromolar, significantly larger than the increase due to backpropagating spikes.The large amplitude, long duration, and unique location of the [Ca²⁺]_i increases due to Ca²⁺ release from stores suggests that these increases can affect specific downstream signaling mechanisms in neurons.     

Ca<Superscript>2+</Superscript>, calmodulin and phospholipids regulate nitricoxide synthase activity in the rabbit submandibular gland

Nitric oxide (NO) plays an important role as an intra- and intercellular signaling molecule in mammalian tissues. In the submandibular gland, NO has been suggested to be involved in the regulation of secretion and in blood flow. NO is produced by activation of NO synthase (NOS). Here, we have investigated the regulation of NOS activity in the rabbit submandibular gland. NOS activity was detected in both the cytosolic and membrane fractions. Characteristics of NOS in the cytosolic and partially purified membrane fractions, such as Km values for l-arginine and EC₅₀ values for calmodulin and Ca²⁺, were similar. A protein band that cross-reacted with anti-nNOS antibody was detected in both the cytosolic and membrane fractions. The membrane-fraction NOS activity increased 1.82-fold with treatment of Triton X-100, but the cytosolic-fraction NOS activity did not. The NOS activity was inhibited by phosphatidic acid (PA) and phosphatidylinositol 4,5-bisphosphate (PIP₂). The inhibitory effects of phospholipids on the NOS activity were relieved by an increase in Ca²⁺ concentrations. These results suggest that the Ca²⁺- and calmodulin-regulating enzyme nNOS occurs in cytosolic and membrane fractions, and PA and PIP₂ regulate the NOS activity in the membrane site by regulating the effect of Ca²⁺ in the rabbit submandibular gland.Communicated by I.D. Hume     

Postsynaptic regulation of synaptic plasticity by synaptotagmin 4 requires both C2 domains

Ca²⁺ influx into synaptic compartments during activity is a key mediator of neuronal plasticity. Although the role of presynaptic Ca²⁺ in triggering vesicle fusion though the Ca²⁺ sensor synaptotagmin 1 (Syt 1) is established, molecular mechanisms that underlie responses to postsynaptic Ca²⁺ influx remain unclear. In this study, we demonstrate that fusion-competent Syt 4 vesicles localize postsynaptically at both neuromuscular junctions (NMJs) and central nervous system synapses in Drosophila melanogaster. Syt 4 messenger RNA and protein expression are strongly regulated by neuronal activity, whereas altered levels of postsynaptic Syt 4 modify synaptic growth and presynaptic release properties. Syt 4 is required for known forms of activity-dependent structural plasticity at NMJs. Synaptic proliferation and retrograde signaling mediated by Syt 4 requires functional C2A and C2B Ca²⁺–binding sites, as well as serine 284, an evolutionarily conserved substitution for a key Ca²⁺-binding aspartic acid found in other synaptotagmins. These data suggest that Syt 4 regulates activity-dependent release of postsynaptic retrograde signals that promote synaptic plasticity, similar to the role of Syt 1 as a Ca²⁺ sensor for presynaptic vesicle fusion.