Removal Of External Calcium Research Articles

An In Situ Kidney Slice Model for Studying Angiotensin II‐ and TRPC5‐Mediated Calcium Signaling

Current approaches to study calcium signaling in glomeruli use isolated glomeruli or cultured cells, however the structure and physiology of the tissue is likely to be perturbed by the isolation process in both models. We used kidney slices maintained in physiological solution to better preserve the macroscopic structure of glomeruli and measured the impact of angiotensin II receptor activation and blockade of Transient Potential Receptor Canonical 5 (TRPC5) to examine their possible interaction in a number of pre‐ and intraglomerular cell types using spinning‐disk confocal calcium imaging.Calcium transients and tissue distortion in intact kidney slices of mice expressing genetically modified calcium‐sensors (GCaMP5) in Cx40‐positive cells were recorded and analyzed using particle analysis.We observed spontaneous calcium transients in preglomerular cells located at the vascular pole that were blocked by the SERCA pump inhibitor, cyclopiazonic acid (CPA). Stimulation with angiotensin II increased calcium activity and contraction in these cells and induced calcium transients in distinct populations of intraglomerular cells that were also blocked with CPA. While removal of external calcium did not change angiotensin II‐induced calcium transients in intraglomerular cells, the frequency of calcium transients in preglomerular cells was significantly reduced. In angiotensin II‐preincubated kidney slices, spontaneous calcium transients in the preglomerular area were diminished by the specific TRPC4/5 inhibitor, ML204. If given alone, ML204 completely inhibited all preglomerular calcium transients.Thick kidney slices maintained in vitro appear to better preserve the structure and responses of different cell types in glomeruli as measured using calcium transients and contractions. In our experimental set‐up, angiotensin II induced calcium transients in intraglomerular cells, while its impact on preglomerular cells appeared more indirect. As the TRPC5 antagonist was more effective at inhibiting calcium transients in preglomerular cells when the tissue was not prestimulated with angiotensin II, we suggest that TRPC5‐mediated calcium signaling is at least party modulated by angiotensin II.Support or Funding InformationSupported by Deutsche Forschungsgemeinschaft (DFG)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Calcium-Dependent and Synapsin-Dependent Pathways for the Presynaptic Actions of BDNF.

We used cultured hippocampal neurons to determine the signaling pathways mediating brain-derived neurotrophic factor (BDNF) regulation of spontaneous glutamate and GABA release. BDNF treatment elevated calcium concentration in presynaptic terminals; this calcium signal reached a peak within 1 min and declined in the sustained presence of BDNF. This BDNF-induced transient rise in presynaptic calcium was reduced by SKF96365, indicating that BDNF causes presynaptic calcium influx via TRPC channels. BDNF treatment increased the frequency of miniature excitatory postsynaptic currents (mEPSCs). This response consisted of two components: a transient component that peaked within 1 min of initiating BDNF application and a second component that was sustained, at a lower mEPSC frequency, for the duration of BDNF application. The initial transient component was greatly reduced by removing external calcium or by treatment with SKF96365, as well as by Pyr3, a selective blocker of TRPC3 channels. In contrast, the sustained component was unaffected in these conditions but was eliminated by U0126, an inhibitor of the MAP kinase (MAPK) pathway, as well as by genetic deletion of synapsins in neurons from a synapsin triple knock-out (TKO) mouse. Thus, two pathways mediate the ability of BDNF to enhance spontaneous glutamate release: the transient component arises from calcium influx through TRPC3 channels, while the sustained component is mediated by MAPK phosphorylation of synapsins. We also examined the ability of these two BDNF-dependent pathways to regulate spontaneous release of the inhibitory neurotransmitter, GABA. BDNF had no effect on the frequency of spontaneous miniature inhibitory postsynaptic currents (mIPSCs) in neurons from wild-type (WT) mice, but surprisingly did increase mIPSC frequency in synapsin TKO mice. This covert BDNF response was blocked by removal of external calcium or by treatment with SKF96365 or Pyr3, indicating that it results from calcium influx mediated by TRPC3 channels. Thus, the BDNF-activated calcium signaling pathway can also enhance spontaneous GABA release, though this effect is suppressed by synapsins under normal physiological conditions.

Sodium influx induced by external calcium chelation decreases human sperm motility

Calcium removal from the medium promptly reduces human sperm motility and induces a Na(+)-dependent depolarization that is accompanied by an increase in intracellular sodium concentration ([Na(+)](i)) and a decrease in intracellular calcium concentration ([Ca(2+)](i)). Sodium loading activates a Na(+)/K(+)-ATPase. Membrane potential (Vm) and [Ca(2+)](i) were simultaneously detected in human sperm populations with the fluorescent probes diSC(3)(5) and fura 2. [Na(+)](i) and was measured independently in a similar fashion using sodium-binding benzofuran isophthalate. Motility was determined in a CASA system, ATP was measured using the luciferin-luciferase assay, and cAMP was measured by radioimmunoassay. Human sperm motility reduction after calcium removal is related to either Na(+)-loading or Na(+)-dependent depolarization, because, under conditions that inhibit the calcium removal-induced Na(+)-dependent depolarization and [Na(+)](i) increase, sperm motility was unaffected. By clamping sperm Vm with valinomycin, we found that the motility reduction associated with the calcium removal was related to sodium loading, and not to membrane potential depolarization. Mibefradil, a calcium channel blocker, markedly inhibited the Na(+)-dependent depolarization and sodium loading, and also preserved sperm motility. In the absence of calcium, both ATP and cAMP concentrations were decreased by 40%. However ATP levels were unchanged when calcium removal was performed under conditions that inhibit the calcium removal-induced Na(+)-dependent depolarization and [Na(+)](i) increase. Human sperm motility arrest induced by external calcium removal is mediated principally by sodium loading, which would stimulate the Na(+)/K(+)-ATPase and in turn deplete the ATP content.

Conductance Properties of the Acetylcholine Receptor Current of Guinea Pig Outer Hair Cells

The nicotinic acetylcholine receptor (AChR) current of outer hair cells (OHCs) was investigated in isolated and voltage-clamped cells under conditions where co-activating Ca(2+)-activated K(+) currents had been abolished using internal BAPTA, external calcium removal and/or depolarisation to positive voltages. The AChR current activated rapidly and thereafter declined in the continued presence of ACh. Reversal potential measurements indicated that it was a non-specific cation current with a substantial Ca(2+) permeability. It had a characteristic bidirectional rectification with an especially prominent outward component in solutions containing 1 mM Ca(2+). The I-V relation was fitted with a single-energy barrier model. The fit suggests a blocking site within the channel, situated about one third of the way through the membrane from the outside and probably normally occupied by Ca(2+) or Mg(2+). The AChR current was sensitive to the external Ca(2+) since it was reduced, to differing extents, in nominally Ca(2+)-free saline or in high Ca(2+) saline (10 mM). In the presence of a nominally Mg(2+)-free solution containing 0.4 mM Ca(2+), the currents were larger, indicating a potentiated response. This type of behaviour is also shown by recombinant α9α10 AChRs, suggesting a close similarity. The AChR current at both positive and negative voltages was reduced in external solutions where most of the Na(+) had been replaced by NMG(+). The conductance properties of the OHC AChR are compared with α9α10 receptors and nicotinic receptors in other hair cells and discussed in terms of the accepted functional role of providing calcium influx leading to efferent synaptic inhibition of hair cells.

A Mutant Connexin50 with Enhanced Hemichannel Function Leads to Cell Death

To determine the consequences of expression of a novel connexin50 (CX50) mutant identified in a child with congenital total cataracts. The GJA8 gene was directly sequenced. Formation of functional channels was assessed by the two-microelectrode voltage-clamp Connexin protein levels and distribution were assessed by immunoblot analysis and immunofluorescence. The proportion of apoptotic cells was determined by flow cytometry. Direct sequencing of the GJA8 gene identified a 137 G>T transition that resulted in the replacement of glycine by valine at position 46 of the coding region of CX50 (CX50G46V). Both CX50 and CX50G46V induced gap junctional currents in pairs of Xenopus oocytes. In single Xenopus oocytes, CX50G46V induced connexin hemichannel currents that were activated by removal of external calcium; their magnitudes were much higher than those in oocytes injected with similar amounts of CX50 cRNA. When expressed in HeLa cells under the control of an inducible promoter, both CX50 and CX50G46V formed gap junctional plaques. Induction of CX50G46V expression led to a decrease in the number of cells and an increase in the proportion of apoptotic cells. CX50G46V-induced cell death was prevented by high concentrations of extracellular calcium ions. Unlike previously characterized CX50 mutants that exhibit impaired trafficking and/or lack of function, CX50G46V traffics properly to the plasma membrane and forms functional hemichannels and gap junction channels; however, it causes cell death even when expressed at minute levels. The biochemical results indirectly suggest a potential novel mechanism by which connexin mutants could lead to cataracts: cytotoxicity due to enhanced hemichannel function.

Phosphorylation Determines the Calmodulin-mediated Ca2+ Response and Water Permeability of AQP0

In Xenopus oocytes, the water permeability of AQP0 (P(f)) increases with removal of external calcium, an effect that is mediated by cytoplasmic calmodulin (CaM) bound to the C terminus of AQP0. To investigate the effects of serine phosphorylation on CaM-mediated Ca(2+) regulation of P(f), we tested the effects of kinase activation, CaM inhibition, and a series of mutations in the C terminus CaM binding site. Calcium regulation of AQP0 P(f) manifests four distinct phenotypes: Group 1, with high P(f) upon removal of external Ca(2+) (wild-type, S229N, R233A, S235A, S235K, K238A, and R241E); Group 2, with high P(f) in elevated (5 mm) external Ca(2+) (S235D and R241A); Group 3, with high P(f) and no Ca(2+) regulation (S229D, S231N, S231D, S235N, and S235N/I236S); and Group 4, with low P(f) and no Ca(2+) regulation (protein kinase A and protein kinase C activators, S229D/S235D and S235N/I236S). Within each group, we tested whether CaM binding mediates the phenotype, as shown previously for wild-type AQP0. In the presence of calmidazolium, a CaM inhibitor, S235D showed high P(f) and no Ca(2+) regulation, suggesting that S235D still binds CaM. Contrarily, S229D showed a decrease in recruitment of CaM, suggesting that S229D is unable to bind CaM. Taken together, our results suggest a model in which CaM acts as an inhibitor of AQP0 P(f). CaM binding is associated with a low P(f) state, and a lack of CaM binding is associated with a high P(f) state. Pathological conditions of inappropriate phosphorylation or calcium/CaM regulation could induce P(f) changes contributing to the development of a cataract.

Intracellular Sodium Increase Induced by External Calcium Removal in Human Sperm

In human sperm, removal of external calcium produces a fast Na(+)-dependent depolarization that is presumably due to sodium permeation through calcium channels. Calcium restoration produces a ouabain-sensitive hyperpolarization that brings the membrane potential to values frequently more negative than resting. In this work, we show evidence indicating that external calcium removal induces an increase in the intracellular sodium ([Na(+)](i)) and that this phenomenon is related to the Na(+)-dependent depolarization. Calcium restoration blocked the [Na(+)](i) increase and then produced a slow decrease that was inhibited by ouabain. The [Na(+)](i) increase was inhibited by nanomolar-micromolar calcium or by millimolar magnesium, which has been previously shown to inhibit the Na(+)-dependent depolarization. This evidence supports the hypothesis that, in zero-calcium medium, a calcium channel that would contribute to resting intracellular calcium levels allows sodium permeation, producing depolarization and a significant [Na(+)](i) increase. Sodium loading would stimulate the Na(+),K(+)-ATPase, the activity of which contributes to the sperm hyperpolarization observed upon calcium restoration.

Roles of Subthreshold Calcium Current and Sodium Current in Spontaneous Firing of Mouse Midbrain Dopamine Neurons

We used a preparation of acutely dissociated neurons to quantify the ionic currents driving the spontaneous firing of substantia nigra pars compacta neurons, isolated from transgenic mice in which the tyrosine hydroxylase promoter drives expression of human placental alkaline phosphatase (PLAP) on the outer surface of the cell membrane. Dissociated neurons identified by fluorescent antibodies to PLAP showed firing properties similar to those of dopaminergic neurons in brain slice, including rhythmic spontaneous firing of broad action potentials and, in some cells, rhythmic oscillatory activity in the presence of tetrodotoxin (TTX). Spontaneous activity in TTX had broader, smaller spikes than normal pacemaking and was stopped by removal of external calcium. Normal pacemaking was also consistently silenced by replacement of external calcium by cobalt and was slowed by more specific calcium channel blockers. Nimodipine produced a slowing of pacemaking frequency. Pacemaking was also slowed by the P/Q-channel blocker omega-Aga-IVA, but the N-type channel blocker omega-conotoxin GVIA had no effect. In voltage-clamp experiments, using records of pacemaking as command voltage, cobalt-sensitive current and TTX-sensitive current were both sizeable at subthreshold voltages between spikes. Cobalt-sensitive current was consistently larger than TTX-sensitive current at interspike voltages from -70 to -50 mV, with TTX-sensitive current larger at voltages positive to -45 mV. These results support previous evidence for a major role of voltage-dependent calcium channels in driving pacemaking of midbrain dopamine neurons and suggest that multiple calcium channel types contribute to this function. The results also show a significant contribution of subthreshold TTX-sensitive sodium current.

Differentiation-dependent alterations in the extracellular ATP-evoked calcium fluxes of cultured skeletal muscle cells from mice

Although extracellular adenosine triphosphate (ATP) has been generally accepted as the regulator of cellular differentiation, the relative contribution of the various purinoreceptor subtypes to purinergic signalling at distinct stages of skeletal muscle differentiation is still poorly understood. Here we measured extracellular ATP-evoked changes in intracellular calcium concentration and surface membrane ionic currents (I (ATP)), calculated the calcium flux (FL) entering the myoplasmic space and compared these parameters at different stages of differentiation on cultured mouse myotubes. The ATP-evoked FL displayed an early peak and then declined to a steady level. With differentiation, the early peak became separated from the maintained component and was absent on mature myotubes. Repeated ATP applications caused desensitization of the response in both immature and differentiated myotubes, owing mainly to the reduction of the early peak of FL in the former and to a decline of both components in the latter group of cells. Depolarization of the cell or removal of external calcium suppressed the early peak. I (ATP) showed no inactivation, and its voltage dependence displayed strong inward rectification. The concentration dependence of I (ATP) can be fitted using a Hill equation, yielding an EC(50) of 56 microM. Results are consistent with the parallel activation of both P2X and P2Y receptors.

Serotonin increases protective duodenal bicarbonate secretion via enteric ganglia and a 5-HT4-dependent pathway

Objective. Serotonin (5-HT) is present in much larger amounts in the gut than in the central nervous system and is predominantly synthesized and stored in mucosal enterochromaffin cells. Bicarbonate secretion by the duodenal mucosa is the major mechanism in maintaining mucosal integrity, neutralizing invading protons within the surface mucus gel. In this study the role of local 5-HT in the control of the protective secretion was investigated. Material and methods. A segment of proximal duodenum was perfused in situ in anaesthetized rats and the alkaline secretion was continuously recorded by pH-stat. Intracellular calcium signalling was measured in clusters of human and rat duodenal enterocytes devoid of neural tissue. After loading with the fluorescent probe, fura-2, the clusters were attached to the bottom of a temperature-controlled perfusion chamber. Results. Close intra-arterial infusion to the duodenal segment of 5-HT (20–200 nmol kg−1 h−1) dose-dependently increased duodenal mucosal HCO3 secretion. A higher dose (2000 nmol kg−1 h−1) did not further increase secretion. Responses were inhibited by the ganglionic blocker and nicotinic receptor antagonist hexamethonium, and were abolished by the 5-HT4 receptor antagonist SB 204070. The 5-HT3 antagonist tropisetron, in contrast, caused only slight inhibition. Viable human and rat duodenal enterocytes responded to 5-HT (100–500 nM) with an increase in intracellular calcium concentration. Pretreatment with SB 204070 or removal of external calcium abolished the response. Conclusions. Stimulation of the duodenal protective secretion by 5-HT thus involves receptors of the 5-HT4 subtype as well as nicotinic transmission, the myenteric plexus being a likely location. In addition, serotonin acts on enterocyte membrane receptors, inducing intracellular calcium signalling.

DrosophilaMushroom Body Kenyon Cells Generate Spontaneous Calcium Transients Mediated by PLTX-Sensitive Calcium Channels

Spontaneous calcium oscillations in mushroom bodies of late stage pupal and adult Drosophila brains have been implicated in memory consolidation during olfactory associative learning. This study explores the cellular mechanisms regulating calcium dynamics in Kenyon cells, principal neurons in mushroom bodies. Fura-2 imaging shows that Kenyon cells cultured from late stage Drosophila pupae generate spontaneous calcium transients in a cell autonomous fashion, at a frequency similar to calcium oscillations in vivo (10-20/h). The expression of calcium transients is up regulated during pupal development. Although the ability to generate transients is a property intrinsic to Kenyon cells, transients can be modulated by bath application of nicotine and GABA. Calcium transients are blocked, and baseline calcium levels reduced, by removal of external calcium, addition of cobalt, or addition of Plectreurys toxin (PLTX), an insect-specific calcium channel antagonist. Transients do not require calcium release from intracellular stores. Whole cell recordings reveal that the majority of voltage-gated calcium channels in Kenyon cells are PLTX-sensitive. Together these data show that influx of calcium through PLTX-sensitive voltage-gated calcium channels mediates spontaneous calcium transients and regulates basal calcium levels in cultured Kenyon cells. The data also suggest that these calcium transients represent cellular events underlying calcium oscillations in the intact mushroom bodies. However, spontaneous calcium transients are not unique to Kenyon cells as they are present in approximately 60% of all cultured central brain neurons. This suggests the calcium transients play a more general role in maturation or function of adult brain neurons.

External bioenergy-induced increases in intracellular free calcium concentrations are mediated by Na+/Ca2+ exchanger and L-type calcium channel

External bioenergy (EBE, energy emitted from a human body) has been shown to increase intracellular calcium concentration ([Ca2+]i, an important factor in signal transduction) and regulate the cellular response to heat stress in cultured human lymphoid Jurkat T cells. In this study, we wanted to elucidate the underlying mechanisms. A bioenergy specialist emitted bioenergy sequentially toward tubes of cultured Jurkat T cells for one 15-minute period in buffers containing different ion compositions or different concentrations of inhibitors. [Ca2+], was measured spectrofluorometrically using the fluorescent probe fura-2. The resting [Ca2+]i in Jurkat T cells was 70 +/- 3 nM (n = 130) in the normal buffer. Removal of external calcium decreased the resting [Ca2+]i to 52 +/- 2 nM (n = 23), indicating that Ca2+ entry from the external source is important for maintaining the basal level of [Ca2+]i. Treatment of Jurkat T cells with EBE for 15 min increased [Ca2+]i by 30 +/- 5% (P < 0.05, Student t-test). The distance between the bioenergy specialist and Jurkat T cells and repetitive treatments of EBE did not attenuate [Ca2+]i responsiveness to EBE. Removal of external Ca2+ or Na+, but not Mg2+, inhibited the EBE-induced increase in [Ca2+]i. Dichlorobenzamil, an inhibitor of Na+/Ca2+ exchangers, also inhibited the EBE-induced increase in [Ca2+]i in a concentration-dependent manner with an IC50 of 0.11 +/- 0.02 nM. When external [K+] was increased from 4.5 mM to 25 mM, EBE decreased [Ca2+]i. The EBE-induced increase was also blocked by verapamil, an L-type voltage-gated Ca2+ channel blocker. These results suggest that the EBE-induced [Ca2+]i increase may serve as an objective means for assessing and validating bioenergy effects and those specialists claiming bioenergy capability. The increase in [Ca2+]i is mediated by activation of Na+/Ca2+ exchangers and opening of L-type voltage-gated Ca2+ channels.

Enhanced response to AVP in the interlobular artery from the spontaneously hypertensive rat

Arginine vasopressin (AVP) induces exaggerated intracellular free calcium (Cai2+) responses in preglomerular smooth muscle cells from young spontaneously hypertensive rats (SHR) due to increased density of the AVP V1a receptor. The intention of the present paper was to examine the relative contribution of afferent arterioles (AA) and interlobular artery (ILA) in AVP- and norepinephrine-induced calcium signaling. The kidneys were perfused with agar solution in vivo, and thin cortical slices were enzyme digested to produce isolated agar-filled vascular fragments. Calcium responses were recorded in fura 2-loaded cells by Ca2+ imaging. Diameter changes were measured after AVP stimulation and mRNA for V1a was measured on isolated vessel fragments. SHR had a significantly higher baseline calcium ratio and lower resting diameter compared with normotensive Wistar-Kyoto rats (WKY). Stimulation with AVP (10(-7) M) in ILA fragments from SHR induced a ratio increase of 0.49 +/- 0.09, significantly higher than the ratio increase in AA from SHR (0.20 +/- 0.03, P < 0.01) and in ILA from WKY (0.24 +/- 0.03, P < 0.01). Stimulation with norepinephrine (10(-7) M) induced responses homogeneously distributed between the segments and strains. Nifedipine treatment or removal of external calcium (Cao2+) reduced the norepinephrine-induced peak response. Both norepinephrine- and AVP-induced sustained responses were abolished after Cao2+ removal in SHR and WKY (P < 0.01). Measurements of V1a receptor mRNA on isolated segments showed a threefold increase in ILA from SHR. The present findings indicate that the exaggerated Ca2+ and contractile response to AVP in SHR is mainly mediated through ILA vasoconstriction.

Determination of depolarisation- and agonist-evoked calcium fluxes on skeletal muscle cells in primary culture

Changes in intracellular calcium concentration ([Ca 2+] i) evoked by prolonged depolarisation (120 mM KCl) or by the application of 15 mM caffeine were measured on skeletal muscle cells in primary culture. The extrusion rate (PV max) of calcium from the myoplasm was determined, which in turn enabled the calculation of the calcium flux (Fl) underlying the measured calcium transients. PV max was found to increase during differentiation, from 107±10 μM/s at the early myotube stage to 596±36 μM/s in secondary myotubes. This was paralleled by a decrease in resting [Ca 2+] i from 99±4 to 51±2 nM. The depolarisation-evoked Fl rose to peak and then ceased despite the continuous presence of KCl. In contrast, the caffeine-induced Fl showed a peak and a clear steady-level with a peak-to-steady ratio of 5.6±1.2. Removal of external calcium suppressed the depolarisation–induced flux by 88±5% indicating that both an influx and a release from the SR underlie the K +-evoked calcium transients. Subsequent applications of caffeine resulted in essentially identical fluxes indicating an efficient refilling of the internal stores. Moreover, if a depolarisation-induced calcium transient preceded the second caffeine-evoked release, the latter was significantly larger than the first suggesting that much of the calcium that entered was stored in the SR rather than extruded.

Polyamine flux in Xenopus oocytes through hemi-gap junctional channels.

Diverse polyamine transport systems have been described in different cells, but the molecular entities that mediate polyamine influx and efflux remain incompletely defined. We have previously demonstrated that spermidine efflux from oocytes is a simple electrodiffusive process, inhibitable by external Ca2+, consistent with permeation through a membrane cation channel. Hemi-gap junctional channels in Xenopus oocytes are formed from connexin 38 (Cx38), and produce a calcium-sensitive (Ic) current that is inhibited by external Ca2+. Spermidine efflux is also calcium sensitive, and removal of external calcium increases both Ic currents and spermidine efflux in Xenopus oocytes. Injection of Cx38 cRNA or Cx38 antisense oligonucleotides (to increase or decrease, respectively, Cx38 expression) also increases or decreases spermidine efflux in parallel. Spermidine efflux has a large voltage-dependent component, which is abolished with injection of Cx38 antisense oligonucleotides. In addition, spermidine uptake is significantly increased in Cx38 cRNA-injected oocytes in the absence of external calcium. The data indicate that hemi-gap junctional channels provide the Ca2+-inhibited pathway for electrodiffusive efflux of polyamines from oocytes, and it is likely that hemi-gap junctional channels provide Ca2+ and metabolism-sensitive polyamine permeation pathways in other cells.

Induction of a Sodium-dependent Depolarization by External Calcium Removal in Human Sperm

Removal of external calcium with EGTA (from 2.5 mm to nanomolar levels) caused a remarkable depolarization in human sperm. This depolarization was initially fast. It was followed by a slow phase that brought the Vm to values of over 0 mV in 1-2 min. The slow and sustained phase correlated with a sustained decrease in intracellular calcium. However, calcium removal still induced depolarization in sperm with enhanced intracellular calcium (induced by progesterone), indicating that the sustained depolarization was not caused by a sustained intracellular calcium decrease. The depolarization was reduced as the external sodium content was substituted with choline, indicating that it was due to a sodium current, and was observed in lithium but not in tetramethylammonium-containing medium. In low sodium medium, the addition of sodium after calcium removal induced depolarization to the extent of which slightly increased in 2 min. The depolarization was completely inhibited by external magnesium (Ki = 1.16 mm). The addition of calcium or magnesium to calcium removal-induced depolarized sperm induced hyperpolarization that was inhibited by ouabain and was also prevented in medium without potassium, suggesting that the activity of the electrogenic Na+,K+-ATPase was involved. The conductance activated by calcium removal might unveil the presence of a calcium channel that in the absence of external calcium allows sodium permeation and that in normal conditions might contribute to the resting intracellular calcium concentration.

Hydrogen peroxide induces loss of dopamine transporter activity: a calcium-dependent oxidative mechanism.

H2O2 dose dependently inhibited dopamine uptake in PC12 cells and in striatal synaptosomes. Treatment with H2O2 resulted in a reversible reduction in Vmax, with no effect on its Km value. This suppressive effect of H2O2 could be relieved by reducing agents (dithiothreitol and cysteine). Furthermore, an oxidizer (dithiodipyridine) also markedly suppressed the dopamine transporter (DAT). Oxidative stress therefore might contribute to the action of H2O2. H2O2 appeared to modify DAT at both extracellular and intracellular sites because cumene-H2O2 (a radical generator mostly restricted to plasma membranes) at high concentrations also slightly suppressed DAT activity and the intracellular overexpression of catalase ameliorated the inhibitory effect of H2O2. Internalization was unlikely to be involved because concanavalin A, which blocked endocytosis, did not prevent the H2O2-evoked inhibition of DAT activity. Interestingly, H2O2 treatment evoked a Ca2+ influx in PC12 cells. Moreover, removal of external calcium by EGTA or reduction in the intracellular calcium level using BAPTA-AM reversed the inhibitory effect of H2O2. Conversely, depletion of intracellular calcium stores using thapsigargin did not affect the reduction in DAT activity by H2O2. Collectively, our results indicate that the DAT, one of the most important proteins controlling the dopaminergic system, is also a redox sensor. In addition, H2O2 might suppress the DAT by a Ca2+-dependent oxidative pathway.

2,5-Anhydro-d-mannitol increases hepatocyte calcium: implications for a hepatic hunger stimulus

The fructose analogue, 2,5-anhydro-d-mannitol (2,5-AM), triggers feeding in rats via a mechanism linked to its ability to trap phosphate and deplete hepatic ATP. This metabolic inhibitor is particularly useful in the study of the role of the liver in initiation of feeding as its effects are preferentially localized to the liver, and its metabolic consequences have been extensively characterized. To determine whether changes in intracellular calcium may participate in a mechanism conveying information about hepatic energy status to the nervous system, we studied the effects of 2,5-AM on intracellular calcium in isolated hepatocytes using the ratiometric indicator, fura-2. 2,5-AM elicited a marked elevation of intracellular calcium within 2–3 min of exposure that returned to baseline upon removal of the agent. Removal of external calcium failed to prevent this response, while emptying intracellular stores prevented it. These data are consistent with the hypothesis that hepatic energy status may be conveyed to the nervous system via a calcium-mediated secretion event.

Vanilloid receptor activation by 2- and 10-μm particles induces responses leading to apoptosis in human airway epithelial cells

Exposure to airborne particulate matter (PM) is associated with increased mortality and morbidity. It has been previously shown that PMs and synthetic particles (PC 10 and PC 2) that have similar characteristics to PMs induced depolarizing currents and increases in intracellular calcium ([Ca 2+] i) in capsaicin- and acid-sensitive sensory neurons and in TRPV1-expressing HEK 293 cells. To determine whether such mechanisms also underlie PM-induced toxicity in epithelial cells lining the human airways, we tested the responses of PCs on BEAS-2B (immortalized human bronchial epithelial cells), NHBE (normal human bronchial/tracheal epithelial cells), and SAEC (normal human small airway epithelial cells from the distal airways). RT-PCR revealed that all these cell types expressed TRPV1 (VR1), ASIC1a, and ASIC3 subunits of proton-gated ion channels. Calcium imaging studies revealed that in all three cell types ∼30% were activated by both capsaicin and acid. In these cells, PCs induced an increase in [Ca 2+] i that was inhibited by capsazepine, a TRPV1 antagonist, and/or by amiloride, an ASIC antagonist. The capsazepine-sensitive contribution to PC-induced increases in [Ca 2+] i was ∼70%. Measurements of apoptosis revealed that exposure to PCs induced a time-dependent increase in the number of apoptotic cells. After incubation for 24 (PC 10) or 48 h (PC 2) ∼60% of these cells were apoptotic. Pretreatment with capsazepine as well as removal of external calcium completely (∼100%) prevented PC-induced apoptosis. These data suggest that pharmacological inhibition of calcium-permeable vanilloid receptors could be used to prevent some of the pathological actions of PMs.

Feline interstitial cystitis results in mechanical hypersensitivity and altered ATP release from bladder urothelium.

ATP can be released from a variety of cell types by mechanical stimulation; however, the mechanism for this release and the influence of pathology are not well understood. The present study examined intracellular signaling mechanisms involved in swelling-evoked (exposure to a hypotonic solution) release of ATP in urothelial cells from normal cats and cats diagnosed with interstitial cystitis (feline interstitial cystitis; FIC). Using the luciferin-luciferase bioluminescent assay, we demonstrate that swelling-evoked ATP release is significantly elevated in FIC cells. In both normal and FIC cells, ATP release was significantly decreased (mean 70% decrease) by application of blockers of stretch-activated channels (amiloride or gadolinium), as well as brefeldin A and monensin (mean 90% decrease), suggesting that ATP release occurs when ATP-containing vesicles fuse with the plasma membrane. Swelling-evoked release was reduced after removal of external calcium (65%), and release was blocked by incubation with BAPTA-AM or agents that interfere with internal calcium stores (caffeine, ryanodine, heparin, or 2-aminoethoxydiphenyl borate). In addition, agents known to act through inositol 1,4,5-triphosphate (IP3) receptors (thapsigargin, acetylcholine) release significantly more ATP in FIC compared with normal urothelium. Taken together, these results suggest that FIC results in a novel hypersensitivity to mechanical stimuli that may involve alterations in IP3-sensitive pathways.