Involvement of intracellular calcium stores in Giardia lamblia induced diarrhoea in mice

Abstract The transmucosal fluxes of Na⁺ and Cl⁻ were studied in Giardia lamblia -infected mice in the presence of absence of dantrolene (1-(5( p -nitrophenyl)furfurilidene-amino) hydantoin sodiumhydrate ). There was net secretion of Na⁺ and Cl⁻ in infected animals, while in control animals there was net absorption of these ions. The addition of dantrolene resulted in significant net increase in absorption of Na⁺ and Cl⁻ in control and experimental groups. Further, mouse intestinal epithelial cells were labelled with [³²P]Pi and then treated with G. lamblia trophozoites and their excretory secretory products separately. The optimum time for inositol triphosphate formation was 15 min in control enterocytes as well as in treated enterocytes. A plateau was formed at higher concentrations. Since raised inositol triphosphate levels mobilize Ca²⁺ from intracellular stores and dantrolene traps Ca²⁺ within intracellular calcium stores, the present study thus suggests that intracellular calcium stores are involved in G. lamblia -induced diarrhoea in mice.     

Comparison of ion transportation before and after egg hatching in Amphinemura sp. (Plecoptera)

Abstract.  An increase in egg size with embryonic development in stoneflies is believed to result from the uptake of water by osmosis. The present study aims to investigate whether a selective ion transport through egg membranes exists before hatching, and whether ions are released after hatching. Viable and nonviable egg masses are incubated in Petri dishes filled with water, and the concentrations of the ions F⁻, Cl⁻, SO₄²⁻, NO₃⁻, Na⁺, K⁻, Mg²⁺ and Ca²⁺ in the water are determined. The ion transport of an egg mass before and after hatching and a nonviable egg mass is then calculated. Before hatching, Cl⁻, SO₄²⁻, NO₃⁻, Na⁺, Mg²⁺ and Ca²⁺ are taken up from the surrounding water into the inner egg. These ions are selectively taken into the egg. After hatching, Cl⁻, SO₄²⁻, Na⁺, Mg²⁺ and Ca²⁺ are released into the surrounding water. The amount of these ions released after hatching is lower than the amount taken up before hatching. Ions that are not released after hatching are considered to be used in embryonic development.     

Signaling Pathway Downstream of GABAA Receptor in the Growth Cone

Abstract: The growth cone is responsible for axonal elongation and pathfinding by responding to various modulators for neurite growth, including neurotransmitters, although the sensor mechanisms are not fully understood. Among neurotransmitters, GABA is most likely to demonstrate activity in vivo because GABA and the GABA_A receptor appear even in early stages of CNS development. We investigated the GABA_A receptor-mediated signaling pathway in the growth cone using isolated growth cones (IGCs). Both the GABA_A binding site and the benzodiazepine modulatory site were enriched in the growth cone membrane. In the intact IGC, GABA induced picrotoxin-sensitive Cl⁻ flux (not influx but efflux) and increased the intracellular Ca²⁺ concentration in a picrotoxin- and verapamil-sensitive manner. Protein kinase C (PKC)-dependent phosphorylation of two proteins identified as GAP-43 and MARCKS protein was enhanced in the intact IGC stimulated by GABA, resulting in the release of MARCKS protein and GAP-43 from the membrane. Collectively, our results suggest the following scheme: activation of the functional GABA_A receptor localized in the growth cone membrane → Cl⁻ efflux induction through the GABA_A-associated Cl⁻ channel → Ca²⁺ influx through an L-type voltage-sensitive Ca²⁺ channel → Ca²⁺-dependent phosphorylation of GAP-43 and MARCKS protein by PKC.     

Mechanisms of GABA- and Glycine-Induced Increases of Cytosolic Ca2+ Concentrations in Chick Embryo Ciliary Ganglion Cells

Abstract: We used fura-2 microfluorometry and the gramicidin-perforated patch clamp technique in an attempt to clarify the mechanisms underlying the GABA-and glycine-induced increases in the cytosolic Ca²⁺ concentration ([Ca]_in) in acutely isolated chick embryo ciliary ganglion neurons. GABA, glycine, and isoguvacine, but not baclofen, increased [Ca]_in in a dose- and a Ca²⁺-dependent manner. The GABA-induced [Ca]_in increase was inhibited by bicuculline and picrotoxin, and potentiated by pentobarbital, flunitrazepam, and alphaxalone, whereas the glycine-induced [Ca]_in increase was inhibited by strychnine but not by bicuculline or picrotoxin. L-and N-type Ca²⁺ channel blockers inhibited the GABA-and glycine-induced [Ca]_in increases, whereas Bay K-8644 potentiated these responses. These responses were also substantially potentiated by blockers of various K⁺ channels and by lowering the external Cl⁻ concentrations. The high KCI- and nicotine-induced [Ca]_in increases were substantially reduced during continuous stimulation with either 2 µ M GABA or 1 m M glycine. Electrophysiological studies indicated that the reversal potential of the GABA-induced current exhibited a more depolarized value than the resting membrane potential in 17 of the 25 cells examined. Taken together, these results suggest that both GABA and glycine depolarize the membrane potentials by increasing Cl⁻ conductance via respective receptors and thus increase the Ca²⁺ influxes through L- and N-type voltage-dependent Ca²⁺ channels.     

Renal excretion in channel catfish following injection of quinaldine sulphate or 3- trifluoromethyl-4-nitrophenol

Channel catfish, Ictalurus punctatus Rafinesque, injected intraperitoneally with 2-methyl-quinoline sulphate (QdSO₄) or 3-trifluoromethyl-4-nitrophenol (TFM) eliminate most of the dose of these compounds by extra-renal routes. Patterns of renal excretion of Na⁺, K⁺, Ca²⁺, Mg²⁺, and Cl⁻ (ρEq kg⁻¹ h⁻¹) appeared to be associated with the 'stress' of the urine collection technique rather than with the elimination of either compound. Concentrations of Na⁺, K⁺, Ca²⁺, Mg²⁺, and Cl⁻ (mEq/1) were determined in urine, plasma and gall bladder bile.     

Loss of ionic and osmotic regulation in Chara internodal cells in concentrated KCl solutions

Intact internodal cells of Chara are known to maintain their osmotic pressures at constant levels in artificial pond water at room temperature. Cell fragments with osmotic pressures higher and those cell fragments with osmotic pressures lower than the original, both of which are prepared from intact internodal cells using transcellular osmosis and ligation with threads, can also return their osmotic pressures to the original level within a week in artificial pond water. These regulatory phenomena are realized mainly by extrusion of K⁺ and Cl⁻ in the cytoplasm and/or vacuole or by absorption of K⁺ and Cl⁻ from the external solution. According to the electrochemical potential difference calculated for K⁺ between the vacuole and the external solution, the cells should be able to maintain these regulatory functions even in 50–100 m M KCl+ 1 m M CaCl₂ solutions. However, novel phenomena were observed when they were immersed in such concentrated KCl solutions. To maintain electroneutrality, their osmotic pressures increased up to ca l MPa in 2 days due to absorption of K⁺ and Cl⁻ and many gradually died over time. Ionic and osmotic reguratory functions of Chara cells were lost when they were immersed in 50–100 m M K-salt solutions containing 1 m M Ca²⁺.     

Apoplastic pH and inorganic ion levels in tomato fruit: A potential means for regulation of cell wall metabolism during ripening

Apoplastic pH and ionic conditions exert strong influence on cell wall metabolism of many plant tissues; however, the nature of the apoplastic environment of ripening fruit has been the subject of relatively few studies. In this report, a pressure-bomb technique was used to extract apoplastic fluid from tomato fruit ( Lycopersicon esculentum Mill.) pericarp at several developmental stages. pH and the levels of K⁺, Na⁺, Ca²⁺, Mg²⁺, Cl⁻ and P were determined and compared with the values for the bulk pericarp and locule tissues. The pH of the apoplastic fluid from pericarp tissue decreased from 6.7 in immature and mature-green fruits to 4.4 in fully-ripe fruit. During the same period, the K⁺ concentration increased from 13 to 37 m M . The levels of Na⁺ and divalent cations did not change, whereas the anions P and Cl⁻ increased in ripe fruit. Ca²⁺ levels remained relatively constant during ripening at 4–5 m M , concentrations that effectively limit pectin solubilization. The electrical conductivity of the apoplastic liquid increased 3-fold during ripening, whereas osmotically active solutes increased 2-fold. Pressure-treated fruit retained the capacity to ripen. The decline in apoplastic pH and increase in ionic strength during tomato fruit ripening may regulate the activity of cell wall hydrolases. The potential role of apoplastic changes in fruit ripening and softening is discussed.     

Germination and seedling growth of cotton: salinity-calcium interactions

Abstract. The effects of NaCl salinity on germination and early seedling growth of cotton were studied. Germination was both delayed and reduced by 200 mol m⁻³ NaCl in the presence of a complete nutrient medium. Seedlings, 7–9 d old, were greatly reduced in fresh weight by salinity. The addition of supplemental Ca²⁺ (10 mol m⁻³ as SO₄²⁻ or Cl⁻) to the medium did not improve germination but, to a large degree, offset the reduction in root growth caused by NaCl. Roots growing in the high salt medium without supplemental Ca²⁺ appeared infected by microbes. The cation specificity of the beneficial Ca²⁺ effect on growth was ascertained by testing additions of MgSO₄ or KCl to the NaCl treatments. The contents of K⁴ and Ca²⁺ were reduced in both roots and shoots by the NaCl treatments. Supplemental Ca²⁺ partially offset this effect for K⁴ in the roots and for Ca²⁺ in both roots and shoots. Sodium contents were not affected by the supplemental Ca²⁺. It is concluded that the beneficial effect of high Ca²⁺ concentrations on root growth of cotton seedlings in a saline environment may be due to maintenance of K/Na-selectivity and adequate Ca status in the root.     

Effects of chloride and sodium on gas exchange parameters and water relations of Citrus plants

Gas exchange parameters, water relations and Na⁺/Cl^- content were measured on leaves of one-year-old sweet orange ( Citrus sinensis [L.] Osbeck cv. Hamlin) seedlings grown at increasing levels of salinity. Different salts (NaCl, KCl and NaNO₃) were used to separate the effects of Cl⁻ and Na⁺ on the investigated parameters. The chloride salts reduced plant dry weight and increased defoliation. Accumulation of Cl⁻ in the leaf tissue caused a sharp reduction in photosynthesis and stomatal conductance. By contrast, these parameters were not affected by leaf Na⁺ concentrations of up to 478 m M in the tissue water. Leaf water potentials reached values near −1.8 MPa at high NaCl and KCl supplies. This reduction was offset by a decrease in the osmotic potential so that turgor was maintained at or above control values. The changes in osmotic potential were closely correlated with changes in leaf proline concentrations. Addition of Ca²⁺ (as calcium acetate) increased growth and halved defoliation of salt stressed plants. Furthermore, calcium acetate decreased the concentration of Cl⁻ and Na⁺ in the leaves, and increased photosynthesis and stomatal conductance. Calcium acetate also counteracted the reductions in leaf water and osmotic potentials induced by salinity. In addition, calcium acetate inhibited the accumulation of proline in the leaves which affected the reduction in osmotic potential. These results indicate that adverse effects of salinity in Citrus leaves are caused by accumulation of chloride.     

CHARACTERIZATION OF HIGH AFFINITY CHOLINE UPTAKE BY TORPEDO CALIFORNICA T-SACS

Abstract— The high affinity choline uptake system present in T-sacs prepared from Torpedo californica electric organ was shown to be insensitive to external Ca²⁺ and to be absolutely dependent on external NaCl, with optimal uptake at approx 200 mM-NaCl. Both Na⁺ and Cl⁻ separately stimulate uptake. Uptake also exhibited an optimum at approx 10mM-external K⁺. Uptake was completely inhibited at 4°C. Approximately 50% of newly accumulated [³H]choline was released by depolarization of T-sacs regardless of the time or method of depolarization.     

Excretion of ions (Cd2+, Li+, Na+ and Cl−) by Tamarix aphylla

Excretion of minerals by the NaCl-resistant and comparatively cadmium-resistant tree Tamarix aphylla (L.) Karst, was investigated. Cd²⁺ was excreted by plants exposed for 1–10 days to 9 or 45 μ M Cd²⁺ solutions. Excretion of this toxic ion increased considerably with time but was less than 5% of the quantities that had been accumulated in the shoots. Excretion of Na⁺ and Cl⁻ was positively correlated with NaCl concentration (1.5, 10, 50 m M ) of the medium. The Na⁺/Cl⁻ ratios of the excrete were positively correlated with the concentration of the treatment solution. Ca²⁺ excretion decreased with increasing NaCl concentrations of the solution. Excretion of K⁺ and Mg²⁺ was only little affected by NaCl. Excretion of Li⁺ occurred whenever this element was supplied in the uptake solution; daily excretion rates of Li⁺ increased with time. The ecological significance of excretion is discussed in relation to the low selectivity of the mechanism in T. aphylla .     

Persistence of the Interaction of Calmodulin with Adenylyl Cyclase: Implications for Integration of Transient Calcium Stimuli

Abstract: Ca²⁺/calmodulin-sensitive adenylyl cyclase plays a role in several forms of synaptic plasticity and learning. To understand how cellular signals from neuronal activity during behavioral stimuli might be integrated by adenylyl cyclase, we have characterized the response of type I adenylyl cyclase to transient Ca²⁺ stimuli. Stimulation by a several second Ca²⁺ stimulus is delayed, rising to a peak after the Ca²⁺ stimulus has ended. We attempted to identify the site of the persistent Ca²⁺ signal that enabled adenylyl cyclase stimulation to increase after free Ca²⁺ had declined. Free calmodulin itself displayed no persistent activation by Ca²⁺ and was unable to activate adenylyl cyclase if exposed to low Ca²⁺ solution <1 s before reaching adenylyl cyclase. In contrast, activation of the calmodulin-adenylyl cyclase complex persisted for seconds after Ca²⁺ stimulus. Activation decayed with a time constant of 6 or 13 s depending on assay conditions. These results suggest that the calmodulin-adenylyl cyclase complex can serve as a site of cellular memory for a Ca²⁺ transient that has ended even before adenylyl cyclase is fully activated.     

The self-incompatibility response in Papaver rhoeas is mediated by cytosolic free calcium

The role of Ca²⁺ signalling during the self-incompatibility (SI) response in Papaver rhoeas L. has been investigated using Ca²⁺-sensitive dyes. Pollen tubes were micro-injected with Calcium Green-1 and cytosolic free calcium ([Ca²⁺]_i) imaged using laser scanning confocal microscopy (LSCM). Addition of incompatible stigmatic S-glycoproteins induced a transient increase in the level of [Ca²⁺]_i in pollen tubes. In contrast, no rise in [Ca²⁺]_i was detectable after addition of either compatible or heat-denatured incompatible stigmatic S-glycoproteins. The elevation of [Ca²⁺]_i was followed by the specific inhibition of pollen tube growth in incompatible reactions. It has been shown previously that gene expression in pollen tubes is switched on during an incompatible reaction. Since the [Ca²⁺]_i transient appeared to originate from the region where the nuclei are located, Ca²⁺ may be involved in locally regulating the expression of these genes. The photoactivation of caged Ca²⁺ to artificially elevate [Ca²⁺]_i resulted in the inhibition of pollen tube growth and thus mimicked the SI response. Taken together, the results provide an important link between a transient rise in [Ca²⁺]_i and the biological phenomenon of inhibition of pollen tube growth and demonstrate, for the first time, direct evidence that the SI response in P. rhoeas is mediated by [Ca²⁺]_i.     

Vascular physiology of a Ca2+ mobilizing second messenger - cyclic ADP-ribose

Cyclic ADP-ribose (cADPR) is a novel Ca²⁺ mobilizing second messenger, which is capable of inducing Ca²⁺ release from the sarcoplasmic reticulum (SR) via activation of ryanodine receptors (RyR) in vascular cells. This signaling nucleotide has also been reported to participate in generation or modulation of intracellular Ca²⁺ sparks ²⁺waves or oscillations, Ca²⁺-induced Ca²⁺ release (CICR) and spontaneous transient outward currents (STOCs) in vascular smooth muscle cells (VSMCs). With respect to the role of cADPR-mediated signaling in mediation of vascular responses to different stimuli, there is accumulating evidence showing that cADPR is importantly involved in the Ca²⁺ response of vascular endothelial cells (ECs) and VSMCs to various chemical factors such as vasoactive agonists acetylcholine, oxotemorine, endothelin, and physical stimuli such as stretch, electrical depolarization and sheer stress. This cADPR-RyR-mediated Ca²⁺ signaling is now recognized as a fundamental mechanism regulating vascular function. Here we reviewed the literature regarding this cADPR signaling pathway in vascular cells with a major focus on the production of cADPR and its physiological roles in the control of vascular tone and vasomotor response. We also summarized some publish results that unveil the underlying mechanisms mediating the actions of cADPR in vascular cells. Given the importance of Ca²⁺ in the regulation of vascular function, the results summarized in this brief review will provide new insights into vascular physiology and circulatory regulation.     

Effects of Calcium Channel Antagonists on Calcium Entry and Glutamate Release from Cultured Rat Cerebellar Granule Cells

Abstract: Using a range of Ca²⁺ channel blockers we have investigated the Ca²⁺ channel subtypes that mediate the depolarisation-induced elevation of the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and glutamate release from cultured rat cerebellar granule cells. ω-Conotoxin-GVIA had little effect on either the transient or plateau phase of the depolarisation-induced [Ca²⁺]_i rise or on glutamate release, ruling out a significant role for N-type Ca²⁺ channels. Nifedipine substantially inhibited the initial transient rise in [Ca²⁺]_i and the plateau phase of the [Ca²⁺]_i rise and glutamate release, suggesting the involvement of L-type Ca²⁺ channels. Both ω-agatoxin and ω-conotoxin-MVIIC also inhibited the transient rise in [Ca²⁺]_i and glutamate release but not the plateau phase of the [Ca²⁺]_i rise. The inhibitions by nifedipine were not increased by coaddition of ω-conotoxin-MVIIC, suggesting overlapping sensitivity to these channel blockers. These data show that glutamate release from granule cells in response to depolarisation with a high KCI level involves Ca²⁺ currents that are sensitive to nifedipine, ω-agatoxin-IVA, and also ω-conotoxin-MVIIC. The overlapping sensitivity of the channels to these toxins prevents attribution of any of the phases of the [Ca²⁺]_i rise or glutamate release to distinct P-, Q-, or O-type Ca²⁺ currents.     

The Wave Pattern of Free Calcium Release Upon Fertilization in Medaka and Sand Dollar Eggs

A transient rise in the concentration of Ca²⁺ in the cortex upon fertilization was demonstrated in medaka eggs injected with aequorin. Detection of the aequorin luminescence with an ultra-high sensitivity photonic microscope system revealed a wave of increased Ca²⁺ concentration starting at the site of sperm entry (animal pole) and being propagated along the cortex of the egg toward the antipode. The wave traversed the entire egg surface within 2–3 min. The peak value of the aequorin luminescence, and therefore the peak value of the Ca²⁺ transient, was generally higher at the site of sperm entry than in other regions. The peak values of the luminescence (and therefore of the Ca²⁺ concentration in the cortex) remained fairly constant during propagation of the wave. Microinjection of Ca²⁺ into the cortex also induced a Ca²⁺ wave. When the egg was stimulated by microinjection of Ca²⁺ at the equatorial region, the Ca²⁺ wave was propagated at a fairly constant speed over the egg surface, except at the region near the vegetal pole where the wave was retarded. Simultaneous recording of the Ca²⁺ wave and the wave of cortical change (breakdown of cortical alveoli) in eggs during fertilization revealed that the Ca²⁺ wave preceded the wave of cortical change.
A Ca²⁺ wave was also demonstrated in sand dollar eggs, although due to their smaller size the phenomenon was not as clear as in medaka eggs.     

Ca2+ UPTAKE BY SYNAPTOSOMES AND ITS EFFECT ON THE INHIBITION OF ACETYLCHOLINE RELEASE BY BOTULINUM TOXIN

Abstract— ⁴⁵Ca²⁺ uptake by cerebral cortex synaptosomes was determined by gel filtration, glass fibre disc filtration under suction and by centrifugation with EGTA present. The filtration methods gave comparable results which were higher than values obtained by the centrifugation method. Uptake was increased by 25mM-K⁺ at all times investigated. The accumulated ⁴⁵Ca²⁺ was bound within the synaptosome. ⁴⁵Ca²⁺-ionophore A23187 stimulated uptake only during the first min; levels of intra-synaptosomal ⁴⁵Ca²⁺ then returned to control values. A23187 also increased intra-synaptosomal Na⁺ and Cl⁻ contents. Botulinum toxin inhibits the K.⁺-stimulated release of [¹⁴C]ACh from synaptosomes but the ionophore released [¹⁴C]ACh from both normal and botulinum-treated preparations in a Ca²⁺-dependent manner. However, it also elicited Ca²⁺-dependent release of [choline. Increased extracellular Ca²⁺ (10 mM and 20 mM) released [¹⁴C]ACh (but not [¹⁴C]choline) from both normal and botulinum-treated synaptosomes. It is concluded that botulinum toxin interferes with the provision of Ca²⁺ essential for the mechanism of ACh release.     

Fast-activating cation channel in barley mesophyll vacuoles. Inhibition by calcium

In contrast to the vacuolar ion channels which are gated open by an increase of cytosolic Ca²⁺ the vacuolar ion currents at resting cytosolic Ca²⁺are poorly explored. Therefore, this study was performed to investigate the properties of the so-called fast-activating vacuolar (FV) current which dominates the electrical characteristics of the tonoplast at physiological free Ca²⁺ concentrations. Patch—clamp measurements were performed on whole barley ( Hordeum vulgare ) mesophyll vacuoles and on excised tonoplast patches. Single ion channels were identified, which, based on their selectivity, activation kinetics, Ca²⁺- and voltage-dependence, carry the whole-vacuole FV current. Reversal potential determinations indicated a K⁺ overs C⁻ permeability ratio of about 30. Both inward and outward whole-vacuole currents as well as the activity of single FV channels were inhibited by an increase of cytosolic Ca²⁺, with a K_d≈ 6 µM. At physiological vacuolar Ca²⁺ activities, the FV channel is an outward-rectifying potassium channel. The FV channel was activated in less than a few milliseconds both by negative and positive potential steps, having a minimal activity that is 40 mV negative of the K⁺ equilibrium potential. It is proposed that transport of K⁺ through this cation channel controls the electrical potential difference across the tonoplast.     

Actin Dynamics Regulates Voltage-Dependent Calcium-Permeable Channels of the Vicia faba Guard Cell Plasma Membrane

Free cytosolic Ca²⁺ ([Ca²⁺]_cyt) is an ubiquitous second messenger in plant cell signaling, and [Ca²⁺]_cyt elevation is associated with Ca²⁺-permeable channels in the plasma membrane and endomembranes regulated by a wide range of stimuli. However, knowledge regarding Ca²⁺ channels and their regulation remains limited in planta . A type of voltage-dependent Ca²⁺-permeable channel was identified and characterized for the Vicia faba L. guard cell plasma membrane by using patch-clamp techniques. These channels are permeable to both Ba²⁺ and Ca²⁺, and their activities can be inhibited by micromolar Gd³⁺. The unitary conductance and the reversal potential of the channels depend on the Ca²⁺ or Ba²⁺ gradients across the plasma membrane. The inward whole-cell Ca²⁺ (Ba²⁺) current, as well as the unitary current amplitude and NP_o of the single Ca²⁺ channel, increase along with the membrane hyperpolarization. Pharmacological experiments suggest that actin dynamics may serve as an upstream regulator of this type of calcium channel of the guard cell plasma membrane. Cytochalasin D, an actin polymerization blocker, activated the NPo of these channels at the single channel level and increased the current amplitude at the whole-cell level. But these channel activations and current increments could be restrained by pretreatment with an F-actin stabilizer, phalloidin. The potential physiological significance of this regulatory mechanism is also discussed.     

Contribution of endoplasmic reticulum to Ca(2+) signals in Dictyostelium depends on extracellular Ca(2+)

We recently reported the first molecular genetic evidence that Dictyostelium Ca²⁺ responses to chemoattractants include a contribution from the endoplasmic reticulum (ER) – responses are enhanced in mutants lacking calreticulin or calnexin, two major Ca²⁺-binding proteins in the ER, even though the influx of Ca²⁺ into the mutants is reduced. Compared with wild-type cells, the ER in the mutants contributes at least 30–70 nM additional Ca²⁺ to the responses. Here we report that this additional ER contribution to the cytosolic Ca²⁺ signal depends upon extracellular Ca²⁺– it does not occur in the absence of extracellular Ca²⁺, increases to a maximum as the extracellular Ca²⁺ levels rise to 10 μM and then remains constant at extracellular Ca²⁺ concentrations up to at least 250 μM. These results suggest that Ca²⁺ influx causes the intracellular release, in the simplest scenario by a mechanism involving Ca²⁺-induced Ca²⁺ release from the ER. By way of contrast, we show that Ca²⁺ responses to mechanical stimulation are reduced, but still occur in the absence of extracellular Ca²⁺. Unlike the responses to chemoattractants, mechanoresponses thus include contributions from the ER that are independent of extracellular Ca²⁺.