Homosynaptic facilitation of transmitter release in crayfish is not affected by mobile calcium chelators: implications for the residual ionized calcium hypothesis from electrophysiological and computational analyses

1. Evoked neurotransmitter release at the crayfish neuromuscular junction was measured in the presence of the cell-permeant calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetotoxymethyl (BAPTA-AM). Excitatory post-synaptic potentials were greatly diminished after application of the intracellular chelator, an effect resulting from attenuation of the rise in the concentration of cytoplasmic Ca2+ ([Ca]i) that is necessary for neurotransmission. However, short-term homosynaptic facilitation of release, the magnitude and time course of which is thought to depend on the accumulation and removal of residual Ca ions (Ca2+), was not affected. Application of the cell-permeant form of ethylene glycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) gave similar results. 2. To interpret these results we developed a reaction-diffusion model in 3D rectangular coordinates for Ca2+ diffusion in the presence of mobile and immobile buffers. Solutions of the model in response to influx of Ca2+ through one or six channels for different diffusion coefficients and no nondiffusable buffer, predict that 1) the time course of residual Ca2+ is very brief, 2) an unrealistically low Ca2+ diffusion coefficient is required for residual calcium, 3) the spatially distributed Ca2+ signal is attenuated by intracellular BAPTA, 4) the rate at which free Ca2+ returns to resting levels, after entry (residual Ca2+) is faster with more mobile buffer, and 5) when pulse trains of Ca2+ channel current are used as input, computed facilitation is comparable to experimental measurements without buffer, but is abolished in the presence of exogenous buffer. 3. When the diffusion coefficient of Ca2+ in water is used, there is no residual Ca2+; however, when 0.1-1.6 mM nondiffusable buffer is present with a fast binding coefficient comparable to BAPTA, there is a very small residual Ca2+ due to the unbinding from the fixed binding sites. The nondiffusable buffer is saturated next to a Ca2+ channel. For this case of the diffusion coefficient of calcium in H2O and nondiffusable buffer, when a moderate amount of diffusable buffer is added to the system containing nondiffusable buffer, the very small residual Ca2+ is substantially reduced. This is because the product of diffusable buffer and Ca2+ is carried away as diffusable product, in contrast to the nondiffusable product releasing Ca2+, after Ca2+ entry ceases. 4. The model predicts that mobile calcium buffers with appropriate physical properties will attenuate facilitation and hasten its decay by removing residual calcium.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of free Ca2+ concentration in hair-cell stereocilia

By affecting the activity of the adaptation motor, Ca2+ entering a hair bundle through mechanoelectrical transduction channels regulates the sensitivity of the bundle to stimulation. For adaptation to set the position of mechanosensitivity of the bundle accurately, the free Ca2+ concentration in stereocilia must be tightly controlled. To define the roles of Ca2+-regulatory mechanisms and thus the factors influencing adaptation motor activity, we used confocal microscopy to detect Ca2+ entry into and clearance from individual stereocilia of hair cells dialyzed with the Ca2+ indicator fluo-3. We also developed a model of stereociliary Ca2+ homeostasis that incorporates four regulatory mechanisms: Ca2+ clearance from the bundle by free diffusion in one dimension, Ca2+ extrusion by pumps, Ca2+ binding to fixed stereociliary buffers, and Ca2+ binding to mobile buffers. To test the success of the model, we compared the predicted profiles of fluo-3 fluorescence during the response to mechanical stimulation with the fluorescence patterns measured in individual stereocilia. The results indicate that all four of the Ca2+ regulatory mechanisms must be included in the model to account for the observed rate of clearance of the ion from the hair bundle. The best fit of the model suggests that a free Ca2+ concentration of a few micromolar is attained near the adaptation motor after transduction-channel opening. The free Ca2+ concentration substantially rises only in the upper portion of the stereocilium and quickly falls toward the resting level as adaptation proceeds.     

Dynamic, spatially nonuniform calcium regulation in frog rods exposed to light

1. In intact rods of the bullfrog, Rana Catesbeiana, that were loaded with Fura-2 by incubation, we made high-resolution measurements of Na:Ca,K exchange currents and measured cytosolic free calcium concentrations during exposure to steps of illumination. The calcium dynamics we observed are indicative of unmanipulated rods because Fura-2 had little effect on calcium buffering within the outer segment. 2. In the dark, the total concentration of calcium within the outer segment, determined by integrating the exchange current, was near 50 microM. The free calcium concentration in darkness was 200-400 nM, indicating that > of = 99% of the internal calcium was bound to buffer molecules or equivalently sequestered. 3. During saturating illumination, the concentration of free calcium near the membrane (assayed by the exchange current) fell more rapidly than the space-averaged free calcium concentration (measured with Fura-2), but both had time courses that were best described by a sum of three exponential terms. The time constants were the same for each assay, but the weighting factors were different. 4. The relationship between the exchange current and space-averaged calcium concentration is consistent with significant concentration gradients within the outer segment resulting from high buffering power, diffusional restrictions, and the fact that all net gain and loss of calcium occurs at the membrane. The data further indicate that effective buffering, and hence calcium mobility, is not uniform within the outer segment. 5. Calcium kinetics were independent of the calcium concentration, indicating that the dominant buffers effectively have a low affinity for calcium (KD > [Ca2+]dark free). 6. The dynamics of calcium changes and of exchange currents evoked by saturating and nonsaturating illumination are completely predictable from changes in the circulating current. Calcium and current are related by a linear transformation, indicating that calcium fluxes within the outer segment are passive and that buffers equilibrate rapidly. 7. Although calcium concentrations change slowly with respect to changes in the circulating current, both measured and calculated calcium dynamics are well correlated with changes in light adaptation. Responses to test flashes depended weakly on the detailed time course of the adapting stimulus but strongly on the free cytosolic calcium concentration at the time the test flash was delivered.     

Analytical steady-state solution to the rapid buffering approximation near an open Ca2+ channel

We derive an analytical steady-state solution for the Ca2+ profile near an open Ca2+ channel based on a transport equation which describes the buffered diffusion of Ca2+ in the presence of rapid stationary and mobile Ca2+ buffers (Wagner and Keizer, 1994). This steady-state rapid buffering approximation gives an upper bound on local Ca2+ elevations such as Ca2+ puffs or sparks when conditions for the validity of the rapid buffering approximation are met and is an alternative to approximations that assume that mobile buffers are unsaturable. This result also provides an analytical estimate of the cytosolic Ca2+ domain concentration ([Ca2+]d) near a channel pore and shows the dependence of [Ca2+]d on moderate concentrations of endogenous mobile buffer, Ca2+ indicator dye, and bulk cytosolic Ca2+. Assuming a simple relationship between [Ca2+]d and the lumenal depletion domain of an intracellular Ca2+ channel, lumenal and cytosolic Ca2+ profiles are matched to give an implicit analytical expression for the effect of bulk lumenal Ca2+ on [Ca2+]d.     

Role of membrane-bound Ca in ghost permeability to Na and K

The permeability of red cell ghosts to K is determined by the amount of membrane-bound Mg which, in turn, depends on internal Mg. Contrasting with such effect, an increase in cellular Ca raises K permeability. To test whether this action is due to a competitive displacement of membrane Mg, the free Ca content of human red cell ghosts was altered by means of Ca-EGTA buffers. Net Na and K movements as well as Ca and Mg bindings were assessed after incubation in a Na-medium at 37 degrees C. Raising Ca from 3 X 10(-7) to 1 X 10(-2) M caused a large K efflux with very little Na gain. Under similar conditions, Ca binding was increased without affecting membrane-bound Mg. Both Ca binding and K loss were markedly diminished by either adding ATP to the hemolytic medium or increasing internal Mg at a fixed Ca concentration. A Scatchard analysis showed three Ca binding sites, two of them having high affinity. It is concluded that Ca action does not arise from a displacement of membrane-bound Mg but from binding to different sites in the membrane. Presumably, high affinity sites are involved in the control of K permeability.     

Attenuation of myocardial reperfusion injury by reducing intracellular calcium overloading with dihydropyridines

The effects of three different dihydropyridine (DHP) calcium channel antagonists, nisoldipine, nimodipine, and nifedipine, on myocardial ischemic and reperfusion injury were studied using isolated rat hearts subjected to ischemia and reperfusion. Hearts were perfused with Krebs-Henseleit bicarbonate buffer containing 0, 4, 16, 64 and 100 nM concentrations of the above dihydropyridines for 15 min. Global ischemia was then induced by terminating the aortic flow for 30 min at 37 degrees, followed by 30 min of reperfusion. Left ventricular (LV) functional (LV developed pressure, its first derivative and coronary flow) and biochemical parameters (creatine kinase release) were monitored prior to ischemia and during reperfusion. In separate group of hearts, intracellular free Ca2+ ([Ca2+]i) was monitored with an intracellular calcium analyzer using a fluorescent Ca2+ indicator (Fura-2 AM). Tissue Ca2+ was also measured by atomic absorption spectroscopy after perfusing the hearts with ion-free cold buffer to wash out extracellular Ca2+. Significant recovery of the coronary flow was observed in all hearts treated with a high concentration (100 nM) of DHPs compared with the control group (P < 0.05), while a lower dose of nisoldipine (16 nM) and nifedipine (64 nM) also improved the coronary flow effectively. Reduction of myocardial creatine kinase release and improvement of the recovery of LV developed pressure, dp/dtmax, were achieved by DHPs in a concentration-dependent manner. A higher concentration of DHPs also decreased the formation of myocardial thiobarbituric acid reactive substances, although these compounds did not possess direct free radical scavenging effects in vitro. Tissue Ca2+ content was reduced significantly in treated groups. The rise of [Ca2+]i during ischemia and reperfusion appeared to be attenuated by these DHPs. The concentration-response study of the three DHPs showed the effective concentrations for reducing [Ca2+]i to be 16, 64 and 100 nM nisoldipine, nifedipine and nimodipine, respectively, in this experimental setting. The above results indicate that pretreatment with DHPs can attenuate the myocardial reperfusion injury by modulating Ca2+ overloading and decreasing the susceptibility of the membrane to free radical attack.     

Arginine vasopressin triggers intracellular calcium release, a calcium-activated potassium current and exocytosis in identified rat corticotropes

Arginine vasopressin (AVP) stimulates the secretion of ACTH from pituitary corticotropes. We investigated the action of AVP in single corticotropes of male rats. Corticotropes were identified with the reverse hemolytic plaque assay using antibodies against ACTH. Using the whole-cell recording technique in conjunction with the fluorescent Ca2+ indicator, indo-1 to measure the concentration of cytosolic free Ca2+ ([Ca2+]i), we show that AVP triggers a transient and plateau pattern of Ca2+ signal. The [Ca2+]i elevation activates the apamin-sensitive Ca2+-activated K+ current, which, in turn, causes membrane hyperpolarization. The Ca2+ signal can be elicited in the absence of extracellular Ca2+ and is mimicked by intracellular inositol 1,4,5-trisphosphate (IP3). Both GDP-beta-S and heparin inhibit the AVP response. Thus, AVP triggers intracellular Ca2+ release from the (IP3)-sensitive store via a GTP binding protein-coupled phosphoinositide pathway. Using the high temporal resolution capacitance measurement to detect exocytosis in single corticotropes, we show that a burst of exocytosis is evoked during the AVP-triggered [Ca2+]i elevation. Exocytosis can also be triggered when Ca2+ is released directly from the IP3-sensitive store via flash photolysis of caged IP3. We conclude that AVP-stimulated ACTH secretion in rat corticotrophs is closely coupled to intracellular Ca2+ release from the IP3-sensitive store.     

Effect of pH and calcium concentration on calcium diffusion in streptococcal model-plaque biofilms

Demineralization during a cariogenic episode is affected by storage and transport in dental plaque of ions released from enamel, and by the effect on both of plaque fluid pH and ion concentrations. To investigate this, 45Ca effusion from a condensed film of streptococci was measured at pH 7, 6 and 5, and 0-20 mmol/l calcium. Cells were loaded into effusion chambers and the appearance of 45Ca and [3H]-inulin in carrier-containing but initially tracer-free buffer was measured. Ratios of 45Ca and [3H]-inulin activity in the initial suspending solution and at equilibrium in the clearance solution, permitted calculation of extracellular volume and bound calcium. The rate of Ca appearance was proportional to the retarded diffusion coefficient (rDe), which was related to the effective diffusion coefficient (De) by: rDe = De/(1 + R) in which R is the ratio of bound to free Ca2+. The rate of Ca2+ effusion increased with calcium concentration, converging on a value of 2.8 x 10(-10) m2/sec. At low pH it reached convergence at a lower [Ca]. This demonstrates that calcium effusion is dependent on binding, so a high proportion of binding sites in plaque will reduce mineral loss in vivo. Loss of binding sites at low pH will increase mineral loss.     

Frequent detection of Kaposi''s sarcoma-associated herpesvirus (human herpesvirus 8) DNA in saliva of human immunodeficiency virus-infected men: clinical and immunologic correlates

Extracellular Ca2+ mediates the cellular and molecular responses to cell stimulation of Chlamydomonas reinhardtii. Extracellular Ca2+ concentrations ([Ca2+]e) must exceed certain threshold values to support flagellar excision by acid shock and to stimulate flagellar outgrowth following mechanical shear of the flagella. Also, the magnitude and duration of flagellar RNA accumulations following acid shock or mechanical shear increase with increasing [Ca2+]e. To better understand the role of Ca2+ in flagellar excision, RNA induction, and outgrowth, we have performed a survey of the ion selectivity of each of these responses to acid shock. We found that flagellar excision in vivo following acid shock was supported by Sr2+ and Ca2+, but no other ion tested. LaCl3 and neomycin prevented flagellar excision following acid shock of cells in Ca2+- or Sr2+-containing buffer. Sr2+ addition to detergent-permeabilized cell models, however, failed to elicit flagellar excision in vitro. Cells failed to regrow flagella following flagellar excision in Sr2+-containing buffer unless exogenous Ca2+ was added. Flagellar RNA accumulations of lower magnitude and shorter duration were measured in cells acid-shocked in Sr2+-containing buffer than in Ca2+-containing buffer. These results demonstrate that a Sr2+ influx can evoke flagellar excision following acid shock, but cannot directly activate the machinery for flagellar excision, suggesting that a Sr2+ influx induces excision by stimulating an intracellular Ca2+ release. Furthermore, they suggest that flagellar outgrowth and normal flagellar RNA induction have a strict requirement for Ca2+, which is not satisfied by the proposed intracellular Ca2+ release.     

Selective transfer of calcium from an acidic compartment to the mitochondrion of Trypanosoma brucei. Measurements with targeted aequorins

Organelle compartments are used by cells as reservoirs of exchangeable Ca2+ and as Ca2+ buffers. The following study uses recombinant aequorins (CYT-AEQ and MT-AEQ) to measure the dynamics of Ca2+ flux between organelles in procyclic forms of the pathogenic protozoan, Trypanosoma brucei. Emphasis is placed on the exchange between an acidic Ca2+ reservoir and the mitochondrion. The mammalian mitochondrial targeting sequence was functional in trypanosomes as determined by immunoblots, immunolocalizations, and the observation that MT-AEQ was in a compartment whose Ca2+ uptake was inhibited 82% with carbonyl cyanide p-trifluoromethoxyphenylhydrazone and KCN. The resting level of free calcium ion concentration in the mitochondrion ([Ca2+]mit) was slightly higher than that in the cytoplasm ([Ca2+]cyt) (400 +/- 50 nM and 290 +/- 40 nM, respectively). Melittin (125 nM) disrupted Ca2+ homeostasis by inducing Ca2+ influx across the plasma membrane. [Ca2+]cyt became slightly elevated to 410 +/- 100 nM, whereas [Ca2+]mit was selectively increased approximately 12-fold, with a broad peak at 4.8 +/- 1.9 microM. At the peak, the mitochondrion contained approximately three times more free Ca2+ than the cytosol. However, mitochondrial retention of the Ca2+ was transient. Similar selective transport into the mitochondrion was observed when Ca2+ efflux from an acidic compartment was induced with monensin (2 microg/ml) in the presence of 5 mM EGTA. [Ca2+]cyt was transiently elevated to 400 +/- 50 nM, whereas [Ca2+]mit was elevated to 3.3+/-1.3 microM. When cells were treated sequentially with monensin (2 microg/ml) and then melittin (200 nM), mitochondrial Ca2+ transport was normal. However, [Ca2+]cyt became elevated to a level that was 1.4-fold higher than with melittin alone. Overall, these data demonstrate that the trypanosome mitochondrion is not a reservoir of exchangeable Ca2+ in the resting cell. However, Ca2+ is selectively channeled to the mitochondrion from the plasma membrane or acidic Ca2+ storage compartment. Additionally, the acidic compartment contributes to maintenance of Ca2+ homeostasis in response to melittin.     

Hydroperoxide-induced increases in intracellular calcium due to annexin VI translocation and inactivation of plasma membrane Ca2+-ATPase

Oxidative stress can cause changes in intracellular free calcium concentration ([Ca2+]i) that resemble those occurring under normal cell signaling. In the alveolar macrophage, hydroperoxide-induced elevation of [Ca2+]i modulates the respiratory burst and other important physiologic functions. The source of Ca2+ released by hydroperoxide is intracellular but separate from the endoplasmic reticulum pool released by receptor-mediated stimuli (Hoyal, C. R., Gozal, E., Zhou, H., Foldenauer, K., and Forman, H. J. (1996) Arch. Biochem. Biophys. 326, 166-171). Previous studies in other cells have suggested that mitochondria are a potential source of oxidant-induced [Ca2+]i elevation. In this study we have identified another potential source of hydroperoxide-releasable intracellular calcium, that bound to annexin VI on the inner surface of the plasma membrane. Translocation of annexin VI from the membrane during exposure to t-butyl hydroperoxide matched elevation of [Ca2+]i as a function of time and t-butyl hydroperoxide concentration. The translocation was possibly due to a combination of ATP depletion and oxidative modification of membrane lipids and proteins. A sustained increase in [Ca2+]i occurring > 50 pmol/10(6) cells (50 microM under these conditions) appeared to be a consequence of membrane Ca2+-ATPase dysfunction. These results suggest that exposure to oxidative stress results in early alterations to the plasma membrane and concomitant release of Ca2+ into the cytosol. In addition it suggests a mechanism for participation of annexin VI translocation that may underlie the alterations in macrophage function by oxidative stress.     

The role of the mitochondria in the clearance of cytosolic calcium

The magnitude and space-temporal profile of the intracellular Ca2+ transients are determined both by the mechanism that decrease and increase calcium levels in the cytoplasm. By the use of cocktails with different content of specific inhibitors of the extrusion and sequester mechanisms, the ability of mitochondrial Ca2+ transport to limit the elevation in free cytosolic Ca2+ concentration, following an imposed Ca2+ load was reexamined, indicating variable data with respect to various cells. In chromaffin cells, inhibition of mitochondrial Ca2+ accumulation with protonophore, dramatically modifies the shape of the [Ca2+]c response, indicating that mitochondrial Ca2+ uptake is an important mechanism for clearance of large Ca2+ loads. By contrast, using digital imaging in the presence of specific mitochondria inhibitors to investigate the [Ca2+]c responses of cerebellar granule cells in which ATP generation has been totally separated from mitochondrial Ca2+ transport, indicates surprising results: it was confirmed that mitochondria in these cells accumulate Ca2+ entering the cell in response to plasma membrane depolarization, but specific abolition of mitochondrial Ca2+ accumulation without ATP depletion significantly decreases the bulk cytoplasmic Ca2+ transients generated by elevated KCl levels, whereas the response in greatly increased when protonophore are present and ATP/ADP ratios are allowed to collapse. The results suggest that nonmitochondrial ATP-dependent transport pathways are primarily responsible for removing excess Ca2+ from the cytoplasm. Far from restricting the elevation in [Ca2+]c in response to a Ca2+ load, functional mitochondria may enhance the elevation in the bulk cytoplasm. The existent conflict of data, suggests the need for a new reevaluation of the role of mitochondria in Ca2+ clearance, and the possibility that mitochondria contribute to, rather than protect against, excitoxicity has to be investigated.     

The effects of raising intracellular calcium on synaptic GABAA receptor-channels

The effects of various calcium (Ca2+) loads imposed through whole-cell patch electrodes on dentate gyrus granule cells were investigated on synaptic GABAA receptor-channels. The kinetics of spontaneous inhibitory postsynaptic currents (sIPSCs) were similar when recorded without any exogenous Ca2+ buffers in the patch electrode or with up to 30 mM BAPTA in the pipette. Unbuffered Ca2+ concentrations of 20-100 microM in the patch pipettes induced a gradual prolongation of miniature IPSC (mIPSC) decays over the course of the recording (10-40 min) with no apparent change in their rise times, peak amplitudes, or frequency of occurrence. This effect was not mimicked by other divalent cations such as strontium. Infusion into the cells of free ionic Ca2+ concentrations buffered with various affinity chelators in the pipette had more pronounced effects on synaptic GABAA currents. Free ionic Ca2+ buffered in the range of 200-400 nM with BAPTA prolonged the decay time constant of mIPSCs. Introducing buffered Ca2+ into the neurons in excess of 1 microM, with a relatively low affinity buffer such as Br2BAPTA, resulted in a marked inhibition of mIPSCs. A similar effect was observed following release of Ca2+ from intracellular stores induced by caffeine (10 mM). We conclude that Ca2+ has a biphasic effect on synaptic GABAA receptor-channels. A high affinity potentiation, consistent with a prolongation of channel burst duration, and a low affinity depression of channel activity both contribute to a complex regulation of synaptic GABAA receptors by [Ca2+]i that has a profound bearing on cellular mechanisms of plasticity and pathological alterations in neuronal excitability.     

Distinct influx pathways, not calcium load, determine neuronal vulnerability to calcium neurotoxicity

Many forms of neurodegeneration are ascribed to excessive cellular Ca2+ loading (Ca2+ hypothesis). We examined quantitatively whether factors other than Ca2+ loading were determinants of excitotoxic neurodegeneration. Cell survival, morphology, free intracellular Ca2+ concentration ([Ca2+]i), and 45Ca2+ accumulation were measured in cultured cortical neurons loaded with known quantities of Ca2+ through distinct transmembrane pathways triggered by excitatory amino acids, cell membrane depolarization, or Ca2+ ionophores. Contrary to the Ca2+ hypothesis, the relationships between Ca2+ load and cell survival, free [Ca2+]i, and Ca2+-induced morphological alterations depended primarily on the route of Ca2+ influx, not the Ca2+ load. Notably, Ca2+ loading via NMDA receptor channels was toxic, whereas identical Ca2+ loads incurred through voltage-sensitive Ca2+ channels were completely innocuous. Furthermore, accounting quantitatively for Ca2+ loading via NMDA receptors uncovered a previously unreported component of L-glutamate neurotoxicity apparently not mediated by ionotropic or metabotropic glutamate receptors. It was synergistic with toxicity attributable to glutamate-evoked Ca2+ loading, and correlated with enhanced cellular ATP depletion. This previously unrecognized toxic action of glutamate constituted a chief excitotoxic mechanism under conditions producing submaximal Ca2+ loading. We conclude that (a) Ca2+ neurotoxicity is a function of the Ca2+ influx pathway, not Ca2+ load, and (b) glutamate toxicity may not be restricted to its actions on glutamate receptors.     

Characterization of a membrane calcium pathway induced by rotavirus infection in cultured cells

Some viruses induce changes in membrane permeability during infection. We have shown previously that the porcine strain of rotavirus, OSU, induced an increase in the permeability to Na+, K+, and Ca2+ during replication in MA104 cells. In this work, we have characterized the divalent cation entry pathway by measuring intracellular Ca2+ in fura-2-loaded MA104 and HT29 cells in suspension. The permeability to Ca2+ and other cations was evaluated by the change of the intracellular concentration following an extracellular cation pulse. Rotavirus infection induced an increase in permeability to Ca2+, Ba2+, Sr2+, Mn2+, and Co2+. The rate of cation entry decreased over time as the intracellular concentration increased during the first 20 s. This indicates that regulatory mechanisms, including channel inactivation, are triggered. La3+ did not enter the cell and blocked the entry of the divalent cations in a dose-dependent manner. Metoxyverapamil (D600), a blocker of L-type voltage-gated channels, partially inhibited the entry of Ca2+ in virus-infected MA104 and HT29 cells. The results suggest that rotavirus infection of cultured cells activates a cation channel rather than nonspecific permeation through the plasma membrane. This activation involves the synthesis of viral proteins through mechanisms yet unknown. The increase in intracellular Ca2+ induced by the activation of this channel may be related to the increase in cytoplasmic and endoplasmic reticulum Ca2+ pools required for virus maturation and cell death.     

Quantitative measurement of calcium flux through muscle and neuronal nicotinic acetylcholine receptors

A new approach was developed to determine quantitatively the fraction of current carried by Ca2+ through an ion channel under physiological conditions. This approach entails the simultaneous measurement of membrane current and intracellular Ca2+ for single cells. Whole-cell patch-clamp techniques were used to measure current, and intracellular Ca2+ was monitored with the fluorescent indicator fura-2. To obtain a quantitative measure of the fraction of current carried by Ca2+, a cell-by-cell calibration method was devised to account for differences among cells in such factors as cellular volume and Ca2+ buffering. The method was used to evaluate the Ca2+ flux through muscle and neuronal nicotinic ACh receptors (nAChRs). In a solution containing 2.5 mM Ca2+ at a holding potential of -50 mV, Ca2+ carries 2.0% of the inward current through muscle nAChRs from BC3H1 cells and 4.1% of the inward current through neuronal nAChRs from adrenal chromaffin cells. The Ca2+ flux through neuronal nAChRs of adrenal chromaffin cells is insensitive to alpha-bungarotoxin. The influx of Ca2+ is voltage dependent, and because of the Ca2+ concentration difference across the cellular membrane, there is Ca2+ influx into the cell even when there is a large net outward current. At both muscle and neuronal cholinergic synapses, activity-dependent Ca2+ influx through nicotinic receptors produces intracellular signals that may have important roles in synaptic development, maintenance, and plasticity.     

Treponema denticola outer membrane inhibits calcium flux in gingival fibroblasts

Treponema denticola is a cultivable oral spirochete which perturbs the cytoskeleton in cultured cells of oral origin, but intracellular signalling pathways by which it affects actin assembly are largely unknown. As the outer membrane (OM) of Treponema denticola disrupts actin-dependent processes that normally require precise control of intracellular calcium, we studied the effects of an OM extract on internal calcium release, ligand-gated and calcium release-activated calcium channels, and related mechanosensitive cation fluxes in human gingival fibroblasts (HGF). Single-cell ratio fluorimetry demonstrated that in resting cells loaded with Fura-2, baseline intracellular Ca2+ concentration ([Ca2+]i) was not affected by treatment with OM extract, but normal spontaneous [Ca2+]i oscillations were dramatically increased in frequency for 20 to 30 min followed by complete blockade. OM extract inhibited ATP-induced and thapsigargin-induced release of calcium from intracellular stores by 40 and 30%, respectively. Addition of Ca2+ to the extracellular pool following depletion of intracellular Ca2+ by thapsigargin and extracellular Ca2+ by EGTA yielded 59% less replenishment of [Ca2+]i in OM extract-treated than in control HGF. In cells loaded with collagen-coated ferric oxide beads to stimulate integrin-dependent calcium release, baseline [Ca2+]i was nearly doubled but was not significantly different in control and OM extract-treated cells. Magnetically generated tensile forces on the beads induced >300% increases of [Ca2+]i above baseline. Cells preincubated with OM extract exhibited dose-dependent and time-dependent reductions in stretch-induced [Ca2+]i transients, which were due to neither loss of beads from the cells nor cell death. The T. denticola OM inhibitory activity was eliminated by heating the OM extract to 60 degrees C and by boiling but not by phenylmethylsulfonyl fluoride treatment. Thus nonlipopolysaccharide, nonchymotrypsin, heat-sensitive protein(s) in T. denticola OM can evidently inhibit both release of calcium from internal stores and uptake of calcium through the plasma membrane, possibly by interference with calcium release-activated channels.     

Interactive Effects of Controlled Drainage and Riparian Buffers on Shallow Groundwater Quality

As a result of recent surface water quality problems in North Carolina, riparian buffers and controlled drainage are being used to reduce the loss of nonpoint source nitrogen from agricultural fields. The effect of controlled drainage and riparian buffers as best management practices to reduce the loss of agricultural nonpoint source nitrogen from the middle coastal plain has not been well documented. The middle coastal plain is characterized by intensive agriculture on sandy soils with deeply incised or channelized streams. A 2-year study was conducted to determine the effectiveness of controlled drainage, riparian buffers, and a combination of both in the middle coastal plain of North Carolina. It was hypothesized that raising the water table near the ditch would enhance nitrate-nitrogen reduction through denitrification. On the sandy soils studied, controlled drainage did not effectively raise the water table near the ditch to a greater degree than observed on the free drainage treatment. Due to random treatment location, the free drainage treatment was installed along a ditch with a shallower impermeable layer compared to the impermeable layer on the controlled drainage treatments (2 m versus 3- to 4-m deep). This resulted in a perched or higher water table on the free drainage treatment. Over 17 storm events, the riparian buffer (free drainage) treatment had an average groundwater table depth of 0.92 m compared to 0.96 and 1.45 m for the combination (riparian buffer and controlled drainage) and controlled drainage treatments, respectively. Nitrate concentration decrease between the field wells and ditch edge wells averaged 29% (buffer only), 63% (buffer and controlled drainage), and 73% (controlled drainage only). Although apparently more nitrate was removed from the groundwater on the controlled drainage treatments, the controlled drainage treatment water table near the ditch was not raised closer to the ground surface compared to the free drainage treatment. Nitrate removal effectiveness was attributed to local soil and landscape properties, such as denitrification in deeper reduced zones of the soil profile.     

Use of fluorescent dyes for measurement and localization of organelles associated with Ca2+ store release in human neutrophils

Fura-2 and its lipid analogue, FFP-18, were used to measure changes in cytosolic free Ca2+ concentration within human neutrophils. Whereas fura-2 was employed to monitor cytosolic Ca2+ increases throughout the cytosol, FFP-18 was used to monitor Ca2+ changes only near the membrane. This latter probe was incorporated into the plasma membrane as its acetoxymethyl ester (FFP-18-AM) but as de-esterification was catalysed by cytosolic esterases, the Ca(2+)-sensing probe (FFP-18 acid) accumulated on the inner face of membrane. The fluorescence of esterified probe on the extracellularly facing membrane leaflet was quenched by the membrane-impermeant ion Ni2+. Under these conditions, near membrane Ca2+ changes which resulted from the release of Ca2+ from intracellular stores was possible by conventional ratio fluorescence measurement of FFP-18. From the timing of arrival of Ca2+ at the plasma membrane, it was proposed that there were two Ca2+ storage sites, liberated by different stimuli, one close to the plasma membrane and the other more distant. In order to discover whether organelles within the neutrophil had distributions which correlate with the Ca2+ release sites, fluorescent dyes for structures within the cytosol were employed. We have previously shown that the location of the intracellular membrane stain, DiOC6 (3) corresponds to the distant Ca2+ release site. Here a second stain, BODIPY-C5 ceramide, has also been used and is shown to stain a peripheral region of the neutrophil, in a similar pattern to the near membrane Ca2+ storage site. These data therefore raise the question of whether these stains mark the organelles in neutrophils which are the two Ca2+ storage and release sites.