Phenylarsine oxide increases intracellular calcium mobility and inhibits Ca(2+)-dependent ATPase activity in thymocytes

A rise in intracellular Ca(2+) levels has been implicated as a regulatory signal for the initiation of lymphocyte proliferation. In the present study the mechanism underlying the elevation of [Ca(2+)] induced by phenylarsine oxide [PAO] was investigated in thymocytes. This agent inhibits HIV-1 replication and also NF-kappaB-mediated activation. It has been reported that the PAO-induced Ca(2+) elevation results from an enhanced plasma membrane calcium permeability in T cells. Here, we present biochemical evidence that the PAO-induced Ca(2+) increase was independent of external Ca(2+). Consistent with these facts, when [Ca(2+)](i) was depleted by prolonged incubation of the cells in Ca(2+)-free medium, PAO addition did not lead to [Ca(2+)](i) increase. These data indicate the involvement of intracellular organelles of thymocytes as the source of Ca(2+). Moreover, evidence is presented that PAO inhibited Ca(2+)-dependent ATPase activity from thymocytes and sarcoplasmic reticulum from skeletal muscle. This inhibition was dose-dependent, with a IC(50) of about 30 microM for both preparations of enzyme. The ability of PAO to inhibit Ca(2+)-dependent ATPase represents a novel mechanism of action for this drug. Present data suggest that the PAO-dependent [Ca(2+)](i) increase could be mainly the result of inhibition of Ca(2+)-dependent ATPase. In addition, we describe also a Ca(2+)-dependence for PAO effect on tyrosine phosphorylation.     

A novel Schistosoma mansoni G protein-coupled receptor is responsive to histamine.

A new cDNA was cloned from the bloodfluke, Schistosoma mansoni and shown to encode a protein with structural characteristics of a biogenic amine G protein-coupled receptor (GPCR). At the amino acid level, the parasite receptor (SmGPCR) shared about the same level of sequence homology (approximately 30%) with all major types of amine GPCRs and could not be identified on the basis of sequence. SmGPCR exhibited several nonconservative substitutions at key GPCR positions, including an unusual asparagine substitution (Asn(111)) for the highly conserved aspartate of transmembrane (TM) 3. The full-length SmGPCR cDNA was double-tagged with N-terminal FLAG and C-terminal hexahistidine epitopes, and was codon-optimized for expression in cultured HEK293 and COS7 cells. In situ immunofluorescence analyses targeting the two N- and C-terminal epitopes demonstrated that the modified SmGPCR was expressed at high level in mammalian cells and assumed a typical GPCR topology, the N-terminus being extracellular and the C-terminus intracellular. Functional activity assays revealed that SmGPCR was responsive to histamine, which caused a dose-dependent elevation in intracellular Ca2+ (EC50=0.54+/-0.05 microM). An Asn(111)-->Asp mutation had no effect on the responsiveness to histamine, suggesting that SmGPCR does not require the TM3 aspartate for agonist activation, in contrast to most amine GPCRs. None of the other monoamines tested had any significant effect on receptor activity, using assays that measured both Ca2+- and cAMP-mediated signaling. The results suggest that SmGPCR is a novel structural class of histamine receptor that may be unique to flatworms.     

A penta-substituted pyridine alkaloid from the rhizome of Jatropha elliptica (Pohl) Muell. Arg. is active against Schistosoma mansoni and Biomphalaria glabrata

Jatropha elliptica is a shrub distributed throughout the north and west of Brazil and reputedly possesses a wide range of therapeutical properties. The roots of this plant possess molluscicidal activity and contain terpenoids, coumarin, lignoid, steroids and alkaloid. In the present study, we assessed the schistosomicidal, miracicidal and cercaricidal activities (against Schistosoma mansoni) and molluscicidal activities (against adults and egg masses of Biomphalaria glabrata) of the alkaloid diethyl 4-phenyl-2,6-dimethyl-3,5-pyridinedicarboxylate, isolated from the ethanol extract of the rhizome of J. elliptica, have been determined. The alkaloid was 100 % lethal to adult schistosomes within 4 days at a concentration of 50 μg/mL. Alterations were observed in the schistosome tegument occasioned by treatment with the alkaloid, such as formation of vesicles and vacuolisation. The extent of tegumental damage of the worm was proportional to the time of incubation and to the concentration of compound. The alkaloid also exhibited a potent cercaricidal activity (LC₁₀₀?=?2 μg/mL); it was totally ineffective against miracicidal forms of the parasite. Moreover, the alkaloid presented strong activity against adult snails (LC₉₀?=?36.43 μg/mL) but was inactive against their egg masses. It is observed then the potential of this compound for the development of new therapies for the treatment of schistosomiasis.     

A comparative study of the Ca2+-Mg2+ dependent ATPase from skeletal muscles of young, adult and old rats

Sarcoplasmic reticulum (SR) vesicles isolated from skeletal muscle of Sprague-Dawley rats ranging in age from 4 months to 28 months were studied and compared. A marked decline, with age, was observed in the amount of (total) SR proteins isolated per gram of muscle tissue used. This decline is in line with the known loss of muscle fiber mass and size with advancing age; however, whether the magnitudes of these two effects are indeed identical, remains to be studied. In contrast, no analogous age-related change was detected in the amount of SR protein per unit mass of rat cardiac muscle. The calcium contents, per mg protein, in SR vesicles isolated from rats of all age groups studied did not differ significantly, and represented only a small fraction of the total capacity of the vesicles for this cation. This capacity was found to decline at old age and this effect, combined with the age-related decrease in the concentration of SR proteins in the tissue, indicate a significant decline in calcium sequestration ability in old muscle. Both basal (Ca2+ independent) and calcium stimulated ATPase activities were found not to be affected by age. In contrast, the efficiency of Ca2+ transport across the SR membrane, as reflected by the number of calcium ions pumped into the vesicles per ATP cleaved, declined from a value of 0.37 at 3-4 months to 0.15 at 24 months. This change may represent an age-related reduction in the fraction of coupled SR vesicles, possibly due to alterations in the membrane. SR vesicle preparations from both young and old rats displayed strongly biphasic inactivation kinetics when incubated at 37 degrees C. This may reflect the heterogeneity of muscles in the tissue used, or be due to the presence of a mixture of coupled and uncoupled vesicles in the SR preparations. The rate of the first step in the ATPase inactivation, in which about 75% of the activity is lost, was found to be affected by age, the old SR vesicles being markedly more labile than their young counterparts. In contrast, no difference was detected between the inactivation kinetics of young and old ATPase proteins dissolved in Triton X-100 and the inactivation was monophasic down to less than 6% of the original activity. These results indicate that the age-related modifications in the stability of the SR calcium pump system involve the membrane but not the ATPase protein. The inactivation of the SR ATPase is believed to proceed via dissociation of the dimeric enzyme to (unstable) subunits. It is therefore likely that changes in the SR membrane in old muscle render the ATPase more dissociable.     

Ca(2+)-dependent negative control mechanism for Ca(2+)-induced Ca2+ release in crayfish muscle.

The mechanism of termination of Ca(2+)-induced Ca2+ release (CICR) from the sarcoplasmic reticulum has been investigated in voltage clamped cut crayfish muscle fibres loaded with rhod-2. During depolarizing steps evoking calcium current (ICa), Ca2+ release was first activated. Then the release rapidly (tau approximately 6 ms) declined, as evidenced by the rate of change of the intracellular fluorescence signal representing a Ca2+ transient. The rapid termination of release was not accounted for by inactivation of the trigger ICa or depletion of Ca2+ from the SR, since the rate at which release declined was constant under conditions where the rate of ICa inactivation and the amount of Ca2+ released varied widely. Pre-elevations of [Ca2+]i with prepulses or photolysis of caged Ca2+ caused depression of Ca2+ release during a subsequent test pulse. When the rate of ICa onset was varied by applying voltage ramps with different slopes, currents with fast onset elicited larger Ca2+ release than calcium currents with slower onset, even though the amplitude of the currents was the same. These results suggest that a Ca(2+)-dependent negative control mechanism exists which mediates the termination of CICR independently of the duration of the trigger ICa and before significant depletion of Ca2+ in the SR occurs.     

The carboxyl terminus of the epithelial Ca(2+) channel ECaC1 is involved in Ca(2+)-dependent inactivation

The family of epithelial Ca(2+) channels (ECaC) is a unique group of highly Ca(2+)-selective channels consisting of two members, ECaC1 and ECaC2. We used carboxyl terminal truncations and mutants to delineate the molecular determinants of the Ca(2+)-dependent inhibition of ECaC. To this end, rabbit ECaC1 was expressed heterologously with green fluorescent protein (GFP) in human embryonic kidney 293 (HEK293) cells using a bicistronic vector. Deletion of the last 30 amino acids of the carboxyl terminus of ECaC1 (G701X) decreased the Ca(2+) sensitivity significantly. Another critical sequence for Ca(2+)-dependent inactivation of ECaC1 was found upstream in the carboxyl terminus. Analysis of truncations at amino acid 635, 639, 646, 649 and 653 disclosed a critical sequence involved in Ca(2+)-dependent inactivation at positions 650-653. C653X showed decreased Ca(2+) sensitivity, comparable to G701X, while E649X lacked Ca(2+)-dependent inactivation. Interestingly, the number of green fluorescent cells, which is an index of the number of transfected cells, was significantly smaller for cells transfected with truncations shorter than E649 than for cells transfected with wild-type ECaC. However, the expression level of GFP was restored in the presence of the ECaC blocker ruthenium red, suggesting that these truncations resulted in deleterious Ca(2+) influx. In conclusion, we have identified two domains in the carboxyl terminus of ECaC1 that control Ca(2+)-dependent inactivation.     

The effect of tumor necrosis factor (TNF-alpha) on calcium (Ca2+) level.

Following addition TNF-alpha to variety of cells the diversity of action has been observed. TNF-alpha induced rapid elevation of intercellular Ca2+ level in all studied cells. This suggests TNF receptor is coupled to phospholipase C.     

Ca(2+)-independent and Ca(2+)-dependent stimulation of quantal neurosecretion in avian ciliary ganglion neurons.

1. Although it is generally agreed that Ca2+ couples depolarization to the release of neurotransmitters, hypertonic saline and ethanol (ETOH) evoke neurosecretion independent of extracellular Ca2+. One possible explanation is that these agents release Ca2+ from an intracellular store that then stimulates Ca(2+)-dependent neurosecretion. An alternative explanation is that these agents act independently of Ca2+. 2. This work extends previous observations on the action of ETOH and hypertonic solutions (HOSM) on neurons to include effects on [Ca2+]i. We have looked for Ca(2+)-independent or -dependent neurosecretion evoked by these agents in parasympathetic postganglionic neurons dissociated from chick ciliary ganglia and maintained in tissue culture. The change in concentration of free Ca2+ in the micromolar range inside neurons ([Ca2+]i) was measured with indo-1 with the use of a Meridian ACAS 470 laser scanning microspectrophotometer. 3. Elevated concentration of extracellular KCl increased [Ca2+]i and the frequency of quantal events. Also, a twofold increase in osmotic pressure (HOSM) produced a similar increase in quantal release and a significant rise in [Ca2+]i; however, the Ca2+ appeared to come from intracellular stores. 4. In contrast, ETOH stimulated quantal neurosecretion without a measurable change in [Ca2+]i. It appears the alcohol exerts its influence on some stage in the process of exocytosis that is distal to or independent of the site of Ca2+ action. 5. The effects of high [KCl]o and osmotic pressure were occlusive. This is explained in part by the observation that hypertonicity reduced Ca2+ current, but an action on Ca2+ stores is also likely.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of Ca(2+) in the rapid cooling-induced Ca(2+) release from sarcoplasmic reticulum in ferret cardiac muscles

Rapid lowering of the solution temperature (rapid cooling, RC) from 24 to 3°C within 3 s releases considerable amounts of Ca²⁺ from the sarcoplasmic reticulum (SR) in mammalian cardiac muscles. In this study, we investigated the intracellular mechanism of RC-induced Ca²⁺ release, especially the role of Ca²⁺, in ferret ventricular muscle. Saponin-treated skinned trabeculae were placed in a glass capillary, and the amount of Ca²⁺ released from the SR by RC and caffeine (50 mM) was measured with fluo-3. It was estimated that in the presence of ATP about 45% of the Ca²⁺ content in the SR was released by RC. The amount of SR Ca²⁺ released by RC was unchanged by the replacement of ATP by AMP-PCP (a non-hydrolysable ATP analogue and agonist for the ryanodine receptor but not for the Ca²⁺ pump of SR), suggesting that the suppression of the Ca²⁺ pump of SR at low temperature might not be a major mechanism in RC-induced Ca²⁺ release. The free Ca²⁺ concentration of the solution used for triggering RC-induced Ca²⁺ release was estimated to be only about 20 nM with fluo-3 or aequorin. When this solution was applied to the preparation at 3°C, only a small amount of Ca²⁺ was released from SR presumably by the Ca²⁺-induced Ca²⁺ release (CICR) mechanism. Thus, in mammalian cardiac muscles, RC releases a part of the (<50%) stored Ca²⁺ contained in the SR, and the mechanism of RC-induced Ca²⁺ release may differ from that of CICR, which is thought to play a role in frog skeletal muscle fibres that express ryanodine receptors of different types.     

The guanine protein coupled receptor rhodopsin is developmentally regulated in the free-living stages of Schistosoma mansoni

Schistosoma mansoni parasites inhabit three distinct environments including water, intermediate molluscan hosts, and definitive vertebrate hosts. Determining how schistosomes interact with these environments may be one mechanism by which suitable vaccines or novel chemotherapeutic targets will be identified. Towards this end, we describe the identification of a 36-kDa S. mansoni protein that shares extensive sequence similarity to light absorbing rhodopsin guanine protein coupled receptors (GPCRs). This protein, S. mansoni rhodopsin (SmRHO), is the first molecularly characterized GPCR described in schistosomes. Sequence analysis reveals that SmRHO shares extensive phylogenetic conservation among rhodopsins/opsins expressed in water-dwelling invertebrates, possibly indicative of orthology. We demonstrate here that SmRHO is expressed in the free-living, light responsive miracidia and cercaria stages and is down-regulated in the adult, vertebrate residing forms. Moreover, we show that SmRHO is localized to sub-tegumental structures found towards the anterior end of cercariae. As SmRHO may be implicated in schistosome photoreception processes, we have begun a search for additional parasite encoded GPCR super-family members, which may be associated with chemoreception, chemotaxis, and olfaction. Identifying and characterizing new GPCRs may uncover hidden aspects of parasite biology useful towards the development of novel intervention strategies.     

H-2 linked immune response to murine experimental Schistosoma mansoni infections.

Mice of two congenic inbred strains C3H/Sn (H-2k) and C3H.B10 (H-2b) were infected with 100 Schistosoma mansoni cercariae. After the infection, the following parameters for the immunological response were studied: worm burden, mortality, antibody titre, spleen index, eosinophilia, delayed type of hypersensitivity and in vitro response to three S. mansoni antigen preparations. No difference in the worm burden and in the in vitro response to the antigen preparations of adult worm antigen, soluble egg antigens and the egg antigen MSA1, was found. The C3H.B10 mice showed a significantly higher mortality, antibody titre and delayed type of hypersensitivity while the C3H/Sn mice showed asignificantly higher spleen index and eosinophilia. This indicates that the H-2 region influences the course of an acute S. mansoni infection, whereas the susceptibility to the infection seems not to be influenced, as is shown by the worm burden.     

Peptidergic modulation of insect voltage-gated Ca(2+) currents: role of resting Ca(2+) current and protein kinases A and C.

The modulation of voltage-gated Ca(2+) currents in isolated dorsal unpaired median (DUM) neurons of cockroach was investigated using whole cell patch clamp. The neuropeptide neurohormone D (NHD), a member of the adipokinetic hormone family, affected Ca(2+) currents at pico- to nanomolar concentrations. It strongly enhanced currents activating at lower depolarizations, whereas those activating at strong depolarizations were slightly attenuated. The first effect results from upregulation of a previously characterized omega-conotoxin MVIIC- and omega-agatoxin IVA-sensitive "mid/low voltage-activated" (M-LVA) Ca(2+) current. The cAMP-analogue 8-bromo-cAMP, forskolin, and the catalytic subunit of protein kinase A (PKA) mimicked the stimulating action of NHD. In addition, preincubation of neurons with the PKA inhibitor KT 5720 abolished the action of NHD. Thus NHD seems to upregulate the M-LVA current via channel phosphorylation by PKA. Activation of protein kinase C by oleoylacetylglycerol (OAG) mimicked the effect of NHD, and subsequent NHD application only enhanced the current to a moderate extent. On the other hand, inhibition of protein kinase C (PKC) by G? 6976 abolished the NHD effect. These results indicate that also PKC, too, may play a role in the peptidergic modulation of the M-LVA Ca(2+) current. The reduction of Ca(2+) currents in the high-voltage-range is caused by the NHD-induced upregulation of a voltage-independent Ca(2+) resting current, I(Ca,R), which most probably leads to enhanced Ca(2+)-dependent inactivation of voltage-gated Ca(2+) currents. To assess the major consequences of the Ca(2+) current changes, current-clamp investigations were performed. Experiments with iberiotoxin, a specific blocker of BK-type Ca(2+)-dependent K(+) currents, and the M-LVA current-blocking omega-toxins suggested that NHD causes-via increasing Ca(2+)-dependent K(+) currents-a larger hyperpolarization of action potentials. The lowering in the action potential threshold produced by NHD, however, seems to be a direct consequence of the hyperpolarizing shift of the activation curve of total Ca(2+) current resulting from NHD-induced upregulation of the M-LVA current component.     

Activation of BK channels in rat chromaffin cells requires summation of Ca(2+) influx from multiple Ca(2+) channels

Large-conductance Ca(2+) and voltage-dependent K(+) channels (BK channels) in many tissues require high Ca(2+) concentrations for activation and therefore might be expected to be tightly coupled to Ca(2+) channels. However, in most cases, little is known about the relative organization of the BK channels and the Ca(2+) channels involved in their activation. We probed the nature of the organization of BK and Ca(2+) channels in rat chromaffin cells by manipulating Ca(2+) influx through Ca(2+) channels and by altering cellular Ca(2+) buffering using EGTA and bis-(o-aminophenoxy)-N,N,N', N'-tetraacetic acid (BAPTA). The results were analyzed to determine the distance between Ca(2+) and BK channels that would be most consistent with the experimental data. Most BK channels are close enough to Ca(2+) channels to be resistant to the buffering action of millimolar of EGTA, but are far enough to be inhibited by BAPTA. Analysis of the EGTA/BAPTA results suggests that BK channels are at a distance of 50 to 160 nm from Ca(2+) channels. A model that assumes random distribution of Ca(2+) and BK channels fails to account for the observed [Ca(2+)](i) detected by BK channels, suggesting that a specific mechanism may exist to mediate the functional coupling between these channels. Importantly, the effects of EGTA and BAPTA cannot be explained by assuming a one-to-one coupling between Ca(2+) and BK channels. Rather, Ca(2+) influx through a number of Ca(2+) channels appears to act in concert to regulate the behavior of any individual BK channel. Thus differences in BK channel open probabilities may be explained by differences in the extent of Ca(2+) domain overlap at the sites of individual BK channels.     

A novel Ca(2+) influx pathway in mammalian primary sensory neurons is activated by caffeine.

Single-cell microfluorimetry and electrophysiology techniques were used to identify and characterize a novel Ca(2+) influx pathway in adult rabbit vagal sensory neurons. Acutely dissociated nodose ganglion neurons (NGNs) exhibit robust Ca(2+)-induced Ca(2+) release (CICR) that can be triggered by 10 mM caffeine, the classic agonist of CICR. A caffeine-induced increase in cytosolic-free Ca(2+) concentration ([Ca(2+)](i)) is considered diagnostic evidence of the existence of CICR. However, when CICR was disabled through depletion of intracellular Ca(2+) stores or pharmacological blockade of intracellular Ca(2+) release channels (ryanodine receptors), caffeine still elicited a significant rise in [Ca(2+)](i) in approximately 50% of NGNs. The same response was not elicited by pharmacological agents that elevate cyclic nucleotide concentrations. Moreover, extracellular Ca(2+) was obligatory for such caffeine-induced [Ca(2+)](i) rises in this population of NGNs, suggesting that Ca(2+) influx is responsible for this rise. Simultaneous microfluorimetry with whole cell patch-clamp studies showed that caffeine activates an inward current that temporally parallels the rise in [Ca(2+)](i). The inward current had a reversal potential of +8.1 +/- 6.1 (SE) mV (n = 4), a mean peak amplitude of -126 +/- 24 pA (n = 4) at E(m) = -50 mV, and a slope conductance of 1.43 +/- 0.79 nS (n = 4). Estimated EC(50) values for caffeine-induced CICR and for caffeine-activated current were 1.5 and approximately 0.6 mM, respectively. These results indicate that caffeine-induced rises in [Ca(2+)](i), in the presence of extracellular Ca(2+), can no longer be interpreted as unequivocal diagnostic evidence for CICR in neurons. These results also indicate that sensory neurons possess a novel Ca(2+) influx pathway.     

Mechanism of Ca2+ transport by Ca2+-Mg2+-ATPase pump: analysis of major states and pathways

Mechanistic studies of Ca2+ transport by the Ca2+-Mg2+-ATPase of skeletal sarcoplasmic reticulum are reviewed, and a unifying model is proposed. The significant steps in the transport cycle are modeled in terms of occupation and disposition of three binding sites on the enzyme: a) two translocation sites capable of binding to Ca2+ or a charge-stoichiometric amount of alkali cation (M+) or H+, b) an ATP-ADP-binding site, and c) a phosphorylation or phosphate-binding site. The normal transport cycle is characterized as the following sequence of steps: a) binding of two Ca2+ and Mg-ATP to external sites with high affinity and random order, b) enzyme phosphorylation, c) inward translocation of the Ca2+-laden sites, d) Ca2+ release to the sarcoplasmic reticulum lumen and ADP release to the external medium (random order), e) binding of Mg2+ or a charge-stoichiometric amount of K+ plus H+ to the translocators, f) dephosphorylation, g) the return of the K+- and H+-laden translocators to the outside, and h) dissociation of K+ and H+ from the translocator and completion of the cycle with step a. The enzyme is characterized as a Ca2+-K+ plus H+ countertransporter. The K+ plus H+ remove Ca2+ from the inwardly oriented translocator, thereby relieving a product inhibition and increasing the rate of enzyme dephosphorylation.     

Ca(2+)-induced Ca(2+) release activates spontaneous miniature outward currents (SMOCs) in parasympathetic cardiac neurons.

Mudpuppy parasympathetic cardiac neurons exhibit spontaneous miniature outward currents (SMOCs) that are thought to be due to the activation of clusters of large conductance Ca(2+)-activated K(+) channels (BK channels) by localized release of Ca(2+) from internal stores close to the plasma membrane. Perforated-patch whole cell recordings were used to determine whether Ca(2+)-induced Ca(2+) release (CICR) is involved in SMOC generation. We confirmed that BK channels are involved by showing that SMOCs are inhibited by 100 nM iberiotoxin or 500 microM tetraethylammonium (TEA), but not by 100 nM apamin. SMOC frequency is decreased in solutions that contain 0 Ca(2+)/3.6 mM Mg(2+), and also in the presence of 1 microM nifedipine and 3 microM omega-conotoxin GVIA, suggesting that SMOC activation is dependent on calcium influx. However, Ca(2+) influx alone is not sufficient; SMOC activation is also dependent on Ca(2+) release from the caffeine- and ryanodine-sensitive Ca(2+) store, because exposure to 2 mM caffeine consistently caused an increase in SMOC frequency, and 10-100 microM ryanodine altered the configuration of SMOCs and eventually inhibited SMOC activity. Depletion of intracellular Ca(2+) stores by the Ca-ATPase inhibitor cyclopiazonic acid (10 microM) inhibited SMOC activity, even when Ca(2+) influx was not compromised. We also tested the effects of the membrane-permeable Ca(2+) chelators, bis-(o-aminophenoxy)-N,N,N', N'-tetraacetic acid-AM (BAPTA-AM) and EGTA-AM. EGTA-AM (10 microM) caused no inhibition of SMOC activation, whereas 10 microM BAPTA-AM consistently inhibited SMOCs. After SMOCs were completely inhibited by BAPTA, 3 mM caffeine caused SMOC activity to resume. This effect was reversible on removal of caffeine and suggests that the source of Ca(2+) that triggers the internal Ca(2+) release channel is different from the source of Ca(2+) that activates clusters of BK channels. We propose that influx of Ca(2+) through voltage-dependent Ca(2+) channels is required for SMOC generation, but that the influx of Ca(2+) triggers CICR from intracellular stores, which then activates the BK channels responsible for SMOC generation.     

CD28-deficient mice generate an impaired Th2 response to Schistosoma mansoni infection

Engagement of CD28 on T cells provides a co-stimulatory signal necessary for T cell activation and differentiation. Recent findings suggest that priming of T helper (Th)2 cells is more dependent on CD28 activation than Th1 cells. The present study examines whether mice that lack expression of CD28 as a result of gene targeting are capable of generating a Th2 response characteristic during infection with the intravascular trematode parasite Schistosoma mansoni. Mutant and control mice were either inoculated in the footpad with S. mansoni eggs (a potent inducer of a Th2 response) or infected percutaneously with the parasite. Draining lymph nodes (after footpad injection) or spleen cells (after natural infection) were harvested at 12 days and 8 weeks, respectively, and examined for cytokine responses to egg antigens. CD28-deficient mice (−/−) generated diminished egg antigen-driven interleukin (IL)-4 and IL-5 production (by 5- to 17-fold, respectively) compared to CD28-expressing (+/+) littermates. In contrast, lymphocyte proliferation and interferon (IFN)-γ production to egg antigens were equivalent for mutant and control mice. Infected CD28−/− mice also had reduced immunoglobulin secretion. Serum levels of parasite antigen-specific IgG1 and polyclonal IgE were significantly diminished in CD28−/− compared to CD28+/+ mice. Lack of CD28 expression had no effect on granuloma formation around eggs trapped in the liver, but increased susceptibility of mice to primary schistosomiasis infection. These studies indicate that CD28 activation contributes to T cell priming required for generation of a Th2 response to an intravascular dwelling helminth parasite.     

Ca(2+) entry through L-type Ca(2+) channels helps terminate epileptiform activity by activation of a Ca(2+) dependent afterhyperpolarisation in hippocampal CA3

In CA3 neurons of disinhibited hippocampal slice cultures the slow afterhyperpolarisation, following spontaneous epileptiform burst events, was confirmed to be Ca(2+) dependent and mediated by K(+) ions. Apamin, a selective blocker of the SK channels responsible for part of the slow afterhyperpolarisation reduced, but did not abolish, the amplitude of the post-burst afterhyperpolarisation. The result was an increased excitability of individual CA3 cells and the whole CA3 network, as measured by burst duration and burst frequency. Increases in excitability could also be achieved by strongly buffering intracellular Ca(2+) or by minimising Ca(2+) influx into the cell, specifically through L-type (but not N-type) voltage operated Ca(2+) channels. Notably the L-type Ca(2+) channel antagonist, nifedipine, was more effective than apamin at reducing the post-burst afterhyperpolarisation. Nifedipine also caused a greater increase in network excitability as determined from measurements of burst duration and frequency from whole cell and extracellular recordings. N-methyl D-aspartate receptor activation contributed to the depolarisations associated with the epileptiform activity but Ca(2+) entry via this route did not contribute to the activation of the post-burst afterhyperpolarisation.We suggest that Ca(2+) entry through L-type channels during an epileptiform event is selectively coupled to both apamin-sensitive and -insensitive Ca(2+) activated K(+) channels. Our findings have implications for how the route of Ca(2+) entry and subsequent Ca(2+) dynamics can influence network excitability during epileptiform discharges.