Intracellular Ca2+ Homeostasis in Trypomastigotes of Trypanosoma cruzi

ABSTRACT Trypomastigotes of Trypanosoma cruzi maintain an intracellular Ca²⁺ concentration([Ca²⁺]_i) of 64 ± 30 nM. Equilibration of trypomastigotes in an extracellular buffer containing 0.5 mM [Ca²⁺]_o (preloaded cells) increased [Ca²⁺]_i < 20 nM whereas total cell Ca²⁺ increased by 1.5 to 2.0 pmole/cell. This amount of Ca²⁺ would be expected to increase [Ca²⁺]_i to > 10 μM suggesting active sequestration of Ca²⁺. We tested the hypothesis that maintenance of [Ca²⁺]_i involved both the sequestration into intracellular storage sites and extrusion into the extracellular space. Pharmacological probes known to influence [Ca²⁺]_i through well characterized pathways in higher eukaryotic cells were employed. [Ca²⁺], responses in the presence or absence of [Ca²⁺]o were measured to asses the relative contribution of sequestration or extrusion processes in [Ca²⁺]_i homeostasis. In the presence of 0.5 mM [Ca²⁺]_o, the ability of several agents to increase [Ca²⁺]_i was magnified in the order ionomycin ? nigericin > thapsigargin > monensin > valinomycin. In contrast, preloading markedly enhanced the increase in [Ca²⁺], observed only in response to monensin. Manoalide, an inhibitor of phospholipase A₂, enhanced the accumulation of [Ca²⁺]_i due to all agents tested, particularly ionomycin and thapsigargin. Our results suggest that sequestration of [Ca²⁺]_i involved storage sites sensitive to monensin and ionomycin whereas extrusion of Ca²⁺ may involve phospholipase A₂ activity. A Na⁺/Ca²⁺ exchange mechanism did not appear to contribute to Ca²⁺ homeostasis.     

Release of mouse eggs from metaphase arrest by protein synthesis inhibition in the absence of a calcium signal or microtubule assembly

Mouse egg activation, which includes release from meiotic metaphase II arrest, results from fertilization-induced increase in intracellular calcium concentration ([Ca²⁺]_i). However, during egg activation caused by exposure to the protein synthesis inhibitor, cycloheximide, [Ca²⁺]_i did not change. Although eggs fertilized in the presence of microtubule inhibitors remain arrested at metaphase, eggs treated for 32 hr with cycloheximide and the microtubule inhibitor, colcemid, formed nuclei. In untreated eggs aged in culture for 24 hr, the microtubule spindles became deformed. These eggs formed nuclei after exposure to cycloheximide, but not the calcium ionophore A23187. Our results indicate that eggs in which protein synthesis is inhibited are released from metaphase without an increase in [Ca²⁺]_i, and despite disruption of the Spindle. © 1995 Wiley-Liss, Inc.     

Rise of intracellular Ca2+ level causes the decrease of cyclin B1 and Mos in the newt eggs at fertilization

Unfertilized eggs of the newt, Cynops pyrrhogaster, are arrested at the second meiotic metaphase, with activity of the M‐phase promoting factor (MPF) maintained at a high level. After fertilization, the eggs resume the cell cycle, and emit the second polar body. When the change in [Ca²⁺]_i in the fertilized eggs was monitored by aequorin, an early increase in [Ca²⁺]_i was observed 5–10 min after insemination and continued for about 30 sec. A late increase in [Ca²⁺]_i then occurred 10–15 min after fertilization and continued for 30–40 min. The injection of 1,2‐Bis (2 aminophenoxy) ethane‐N,N,N′,N′,‐tetraacetic acid (BAPTA) into unfertilized eggs inhibited reinitiation of the cell cycle after fertilization. Western blot analysis with antibodies against cyclin B1 or Mos indicated that both cyclin B1 and Mos were present in unfertilized eggs, but both disappeared within 30 min after fertilization. Treatment with Ca²⁺‐ionophore decreased both cyclin B1 and Mos. Chymotryptic activity in Cynops egg extracts was not significantly increased after fertilization or activation by treatment with the Ca²⁺‐ionophore. No change in [Ca²⁺]_i was observed following treatment with cycloheximide, but the amount of both cyclin B1 and Mos rapidly decreased. These results indicate that resumption of meiosis in Cynops eggs is induced by an increase in [Ca²⁺]_i at fertilization, which causes degradation of both cyclin B1 and Mos by inhibition of de novo synthesis of those proteins. Mol. Reprod. Dev. 53:341–349, 1999. © 1999 Wiley‐Liss, Inc.     

The extracellular calcium‐sensing receptor promotes porcine egg activation via calcium/calmodulin‐dependent protein kinase II

Extracellular calcium is required for intracellular Ca²⁺ oscillations needed for egg activation, but the regulatory mechanism is still poorly understood. The present study was designed to demonstrate the function of calcium‐sensing receptor (CASR), which could recognize extracellular calcium as first messenger, during porcine egg activation. CASR expression was markedly upregulated following egg activation. Functionally, the addition of CASR agonist NPS R‐568 significantly enhanced pronuclear formation rate, while supplementation of CASR antagonist NPS2390 compromised egg activation. There was no change in NPS R‐568 group compared with control group when the egg activation was performed without extracellular calcium addition. The addition of NPS2390 precluded the activation‐dependent [Ca²⁺]_i rise. When egg activation was conducted in intracellular Ca²⁺ chelator BAPTA‐AM and NPS R‐568 containing medium, CASR function was abolished. Meanwhile, CASR activation increased the level of the [Ca²⁺]_i effector p‐CAMKII, and the presence of KN‐93, an inhibitor of CAMKII, significantly reduced the CASR‐mediated increasement of pronuclear formation rate. Furthermore, the increase of CASR expression following activation was reversed by inhibiting CAMKII activity, supporting a positive feedback loop between CAMKII and CASR. Altogether, these findings provide a new pathway of egg activation about CASR, as the extracellular Ca²⁺ effector, promotes egg activation via its downstream effector and upstream regulator CAMKII.     

Localized calcium signaling in multinucleated osteoclasts

Localized intracellular Ca²⁺ ([Ca²⁺]_i) pulses, fluctuations, and repetitive spikes were detected in multinucleated rabbit osteoclasts in the presence of serum and in response to calcitonin using the fluorescent calcium indicator fluo-3 and a laser scanning microscope. We observed that these [Ca²⁺]_i changes were often restricted within a region of the cell body or propagated from the initial region of occurrence to other parts of the cell body but not to all parts. These observations suggest the existence of significant barriers to Ca²⁺ transport between different cytoplasmic regions of the osteoclast. To further investigate this phenomenon, we mechanically perturbed different cellular regions by touching locally with a micropipette. This usually induced a local increase in cytosolic and nuclear free [Ca²⁺]_i. In some cases there was propagation of the [Ca²⁺]_i increase to other regions but with part of the cell body not affected. Those regions of the cell body to which the [Ca²⁺]_i increase did not propagate had a [Ca²⁺]_i response to a direct mechanical perturbation. Our data show that osteoclasts can have different [Ca²⁺]_i activities in apparently equivalent cellular regions, no matter how generated. This suggests that there can be a number of spatially separate Ca²⁺ regulatory systems within an osteoclast cell body. © 1996 Wiley-Liss, Inc.     

Activation of protein kinase C after fertilization is required for remodeling the mouse egg into the zygote

Fertilization of the mammalian egg initiates numerous biochemical and structural changes which remodel the egg into a single-celled zygote. To date, the most extensively studied phenomenon of fertilization in virtually all species has been the relationship between sperm penetration and the induction of the initial rise in intracellular-free calcium ([Ca²⁺]_i) concentration within the egg. In contrast, relatively few studies have focused on the biochemical events following this rise in calcium, and even fewer studies have directly linked the biochemical events to the structural changes which must ensue for proper development of the embryo. In this study, we exploited recently developed technologies to investigate the action of protein kinase C (PKC), a presumed downstream transducer of the initial rise in [Ca²⁺]_i, during fertilization and artificial activation with calcium ionophore or phorbol 12-myristate 13-acetate (PMA). The newly developed myristoylated PKC pseudosubstrate (myrPKCΨ) was used to specifically inhibit PKC, thereby averting the trauma of injecting the egg with nonmyristoylated PKCΨ. Following fertilization, eggs which were pretreated with myrPKCΨ were not capable of forming a second polar body and pronuclear formation was significantly inhibited. Spatial and temporal localization of PKC using confocal microscopy to visualize the PKC reporter dye, Rim-1, demonstrated localization of PKC to the lateral aspects of the forming second polar body after fertilization, or after artificial activation with calcium ionophore or PMA. In vivo biochemical analysis of eggs which were fertilized or artificially activated demonstrated that PKC activity rose at the same time (40 min) as the second polar body formed and then subsided over the next 5 hr post activation. From these data, we conclude that PKC plays an integral role in directing the transformation from egg to embryo. Mol. Reprod. Dev. 46:587–601, 1997. © 1997 Wiley-Liss, Inc.     

Ca as second messenger in PTTH-stimulated prothoracic glands of the silkworm, Bombyx mori

Measurements of Ca²⁺ influx and [Ca²⁺]_i changes in Fura-2/AM-loaded prothoracic glands (PGs) of the silkworm, Bombyx mori, were used to identify Ca²⁺ as the actual second messenger of the prothoracicotropic hormone (PTTH) of this insect. Dose-dependent increases of [Ca²⁺]_i in PG cells were recorded in the presence of recombinant PTTH (rPTTH) within 5 minutes. The rPTTH-mediated increases of [Ca²⁺]_i levels were dependent on extracellular Ca²⁺. They were not blocked by the dihydropyridine derivative, nitrendipine, an antagonist of high-voltage-activated (HVA) Ca²⁺ channels, and by bepridil, an antagonist of low-voltage-activated (LVA) Ca²⁺ channels. The trivalent cation La³⁺, a non-specific blocker of plasma membrane Ca²⁺ channels, eliminated the rPTTH-stimulated increase of [Ca²⁺]_i levels in PG cells and so did amiloride, an inhibitor of T-type Ca²⁺ channels. Incubation of PG cells with thapsigargin resulted in an increase of [Ca²⁺]_i levels, which was also dependent on extracellular Ca²⁺ and was quenched by amiloride, suggesting the existence of store-operated plasma membrane Ca²⁺ channels, which can also be inhibited by amiloride. Thapsigargin and rPTTH did not operate independently in stimulating increases of [Ca²⁺]_i levels and one agent’s mediated increase of [Ca²⁺]_i was eliminated in the presence of the other. TMB-8, an inhibitor of intracellular Ca²⁺ release from inositol 1,4,5 trisphosphate (IP₃)-sensitive Ca²⁺ stores, blocked the rPTTH-stimulated increases of [Ca²⁺]_i levels, suggesting an involvement of IP₃ in the initiation of the rPTTH signaling cascade, whereas ryanodine did not influence the rPTTH-stimulated increases of [Ca²⁺]_i levels. The combined results indicate the presence of a cross-talk mechanism between the [Ca²⁺]_i levels, filling state of IP₃-sensitive intracellular Ca²⁺ stores and the PTTH-receptor’s-mediated Ca²⁺ influx.     

Spike‐dependent calcium influx in dendrites of the cricket giant interneuron

Identified wind‐sensitive giant interneurons in the cricket's cercal sensory system integrate cercal afferent signals and release an avoidance behavior. A calcium‐imaging technique was applied to the giant interneurons to examine the presence of the voltage‐dependent Ca²⁺ channels (VDCCs) in their dendrites. We found that presynaptic stimuli to the cercal sensory nerve cords elevated the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in the dendrites of the giant interneurons. The dendritic Ca²⁺ rise coincided with the spike burst of the giant interneurons, and the rate of Ca²⁺ rise depended on the frequency of the action potentials. These results suggest that the action potentials directly caused [Ca²⁺]_i increase. Observation of the [Ca²⁺]_i elevation induced by depolarizing current injection demonstrates the presence of the VDCCs in the dendrites. Although hyperpolarizing current injection into the giant interneuron suppressed action potential generation, EPSPs could induce no [Ca²⁺]_i increase. This result means that ligand‐gated channels do not contribute to the synaptically stimulated Ca²⁺ elevation. On the other hand, antidromically stimulated spikes also increased [Ca²⁺]_i in all cellular regions including the dendrites. And bath application of a mixture of Ni²⁺, Co²⁺, and Cd²⁺ or tetrodotoxin inhibited the [Ca²⁺]_i elevation induced by the antidromic stimulation. From these findings, we suppose that the axonal spikes antidromically propagate and induce the Ca²⁺ influx via VDCCs in the dendrites. The spike‐dependent Ca²⁺ elevation may regulate the sensory signals processing via second‐messenger cascades in the giant interneurons. © 2000 John Wiley & Sons, Inc. J Neurobiol 44: 45–56, 2000     

The electrical block to polyspermy induced by an intracellular Ca2+ increase at fertilization of the clawed frogs,Xenopus laevis and Xenopus tropicalis

Polyspermy blocking, to ensure monospermic fertilization, is necessary for normal diploid development in most animals. We have demonstrated here that monospermy in the clawed frog, Xenopus tropicalis, as well as in X. laevis, is ensured by a fast, electrical block to polyspermy on the egg plasma membrane after the entry of the first sperm, which is mediated by the positive‐going fertilization potential. An intracellular Ca²⁺ concentration ([Ca²⁺]_i) at the sperm entry site was propagated as a Ca²⁺ wave over the whole egg cytoplasm. In the X. tropicalis eggs fertilized in 10% Steinberg's solution, the positive‐going fertilization potential of +27 mV was generated by opening of Ca²⁺‐activated Cl^?‐channels (CaCCs). The fertilization was completely inhibited when the egg's membrane potential was clamped at +10 mV and 0 mV in X. tropicalis and X. laevis, respectively. In X. tropicalis, a small number of eggs were fertilized at 0 mV. In the eggs whose membrane potential was clamped below ?10 mV, a large increase in inward current, the fertilization current, was recorded and allowed polyspermy to occur. A small initial step‐like current (IS current) was observed at the beginning of the increase in the fertilization current. As the IS current was elicited soon after a small increase in [Ca²⁺]_i, this is probably mediated by the opening of CaCCs. This study not only characterized the fast and electrical polyspermy in X. tropicalis, but also explained that the initial phase of [Ca²⁺]_i increase causes IS current during the early phase of egg activation of Xenopus fertilization.     

Mechanisms of calcium transport in the giant axon of the squid and their physiological role

The giant axon of the squid has been extensively used as a model for studying Ca regulation in excitable cells. Different techniques (extrusion, injection and dialysis) have been employed to characterize Ca fluxes across the axon membrane. Since both Ca efflux and influx are markedly dependent on [Ca²⁺]_i, considerable effort has been dedicated to determine the resting value of the [Ca²⁺]_i. Results from different laboratories indicate that the [Ca²⁺]_i, in a normal fibre, range from 20–100 nM. Under dialysis conditions (internal control), with an imposed [Ca²⁺]_i of 80 nM, Ca influx is balanced by an outward Ca movement of about 40 f/CS. Ca extrusion occurs through two parallel transport systems: one having a high affinity for [Ca²⁺]_i, dependent on ATP, not affected by Na_i, Na_o, Ca_o, Mg_o and inhibited by internal vanadate (uncoupled component), the other, more prominent at relatively high [Ca²⁺]_i, does not require ATP, is inhibited by Na_i activated by Na_o and not inhibited by vanadate. (Na_o-dependent component). The existence of these two systems provide the axon with an effective way to maintain in the long term a constant low [Ca²⁺]_i in spite of short term fluctuations due to increased Ca influx during nervous activity.     

Inositol 1,4,5‐trisphosphate receptor 1 degradation in mouse eggs and impact on [Ca2+]i oscillations

The initiation of normal embryo development depends on the completion of all events of egg activation. In all species to date, egg activation requires an increase(s) in the intracellular concentration of calcium ([Ca²⁺]_i), which is almost entirely mediated by inositol 1,4,5‐trisphosphate receptor 1 (IP₃R1). In mammalian eggs, fertilization‐induced [Ca²⁺]_i responses exhibit a periodic pattern that are called [Ca²⁺]_i oscillations. These [Ca²⁺]_i oscillations are robust at the beginning of fertilization, which occurs at the second metaphase of meiosis, but wane as zygotes approach the pronuclear stage, time after which in the mouse oscillations cease altogether. Underlying this change in frequency are cellular and biochemical changes associated with egg activation, including degradation of IP₃R1, progression through the cell cycle, and reorganization of intracellular organelles. In this study, we investigated the system requirements for IP₃R1 degradation and examined the impact of the IP₃R1 levels on the pattern of [Ca²⁺]_i oscillations. Using microinjection of IP₃ and of its analogs and conditions that prevent the development of [Ca²⁺]_i oscillations, we show that IP₃R1 degradation requires uniform and persistently elevated levels of IP₃. We also established that progressive degradation of the IP₃R1 results in [Ca²⁺]_i oscillations with diminished periodicity while a near complete depletion of IP₃R1s precludes the initiation of [Ca²⁺]_i oscillations. These results provide insights into the mechanism involved in the generation of [Ca²⁺]_i oscillations in mouse eggs. J. Cell. Physiol. 222:238–247, 2010. © 2009 Wiley‐Liss, Inc.     

Bergmann glial cells in situ express endothelinB receptors linked to cytoplasmic calcium signals

The endothelin (ET) isoforms ET-1, ET-2 and ET-3 applied at 100 nM triggered a transient increase in [Ca²⁺]_i in Bergmann glial cells in cerebellar slices acutely isolated from 20–25 day-old mice. The intracellular calcium concentration ([Ca²⁺]_i) was monitored using Fura-2-based ([Ca²⁺]_i) microfluorimetry. The ET-triggered ([Ca²⁺]_i) transients were mimicked by ET, receptor agonist BO-3020 and were inhibited by ETB receptor antagonist BQ-788. ET elevated [Ca²⁺]_i in Ca2+-free extracellular solution and the ET-triggered [Ca²⁺]_i elevation was blocked by 500 nM thapsigargin indicating that the [Ca²⁺]_i was released from InsP3 sensitive intracellular pools. The ET-triggered [Ca²⁺]_i increase in Ca²⁺-free solution was shorter in duration. Restoration of normal extracellular [Ca²⁺] briefly after the ET application induced a second [Ca²⁺]_i increase indicating the presence of a secondary Ca²⁺ influx which prolongs the Ca²⁺ signal. Pre-application of 100 μM ATP or 10 μM noradrenaline blocked the ET response suggesting the involvement of a common Ca²⁺ depot. The expression of ETB receptor mRNAs in Bergmann glial cells was revealed by single-cell RT-PCR. The mRNA was also found in Purkinje neurones, but no Ca²⁺ signalling was triggered by ET. We conclude that Bergmann glial cells are endowed with functional ET_B receptors which induce the generation of intracellular [Ca²⁺]_i signals by activation of Ca²⁺ release from InsP₃-sensitive intracellular stores followed by a secondary Ca²⁺ influx.     

Dynamic changes of [Ca2+]i in cerebellar granule cells exposed to pulsed electric fields

Intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in embryonic chick cerebellar granule cells loaded with fluo-3/AM and exposed to a single pulsed electric field was investigated using a confocal laser scanning microscope and fluorescent microscope equipped with CCD video imaging system. The results showed that [Ca²⁺]_i increased immediately and rose to the peak rapidly as the cells exposed to a single pulsed electric field. The amplitude and rate of the increases of [Ca²⁺]_i depend on the intensity of external electric field. In the presence of Ca²⁺ chelant EGTA or Ca²⁺ channels blocker La³⁺ in the pulsation solutions, the increase of [Ca²⁺]_i was still observable. It was also observed that [Ca²⁺]_i of different intracellular areas in the cell elevated simultaneously while the peak of the increase of [Ca²⁺]_i in the poles of the cell preceded to the peak in its somata and recovered to a plateau within a short time.     

Mechanotransduction in Colonic Smooth Muscle Cells

We evaluated mechanisms which mediate alterations in intracellular biochemical events in response to transient mechanical stimulation of colonic smooth muscle cells. Cultured myocytes from the circular muscle layer of the rabbit distal colon responded to brief focal mechanical deformation of the plasma membrane with a transient increase in intracellular calcium concentration ([Ca²⁺] _i ) with peak of 422.7 ± 43.8 nm above an average resting [Ca²⁺] _i of 104.8 ± 10.9 nm (n= 57) followed by both rapid and prolonged recovery phases. The peak [Ca²⁺] _i increase was reduced by 50% in the absence of extracellular Ca²⁺, while the prolonged [Ca²⁺] _i recovery was either abolished or reduced to ≤15% of control values. In contrast, no significant effect of gadolinium chloride (100 μm) or lanthanum chloride (25 μm) on either peak transient or prolonged [Ca²⁺] _i recovery was observed. Pretreatment of cells with thapsigargin (1 μm) resulted in a 25% reduction of the mechanically induced peak [Ca²⁺] _i response, while the phospholipase C inhibitor U-73122 had no effect on the [Ca²⁺] _i transient peak. [Ca²⁺] _i transients were abolished when cells previously treated with thapsigargin were mechanically stimulated in Ca²⁺-free solution, or when Ca²⁺ stores were depleted by thapsigargin in Ca²⁺-free solution. Pretreatment with the microfilament disrupting drug cytochalasin D (10 μm) or microinjection of myocytes with an intracellular saline resulted in complete inhibition of the transient. The effect of cytochalasin D was reversible and did not prevent the [Ca²⁺] _i increases in response to thapsigargin. These results suggest a communication, which may be mediated by direct mechanical link via actin filaments, between the plasma membrane and an internal Ca²⁺ store. Received: 24 March 1997/Revised: 21 July 1997     

Effects of the anesthetics heptanol,halothane and isoflurane on gap junction conductance in crayfish septate axons: A calcium- and hydrogen-independent phenomenon potentiated by caffeine and theophylline,and inhibited by 4-aminopyridine

Summary This study has monitored junctional and nonjunctional resistance. [Ca²⁺] _i and [H^–] _i , and the effects of various drugs in crayfish septate axons exposed to neutral anesthetics. The uncoupling efficiency of heptanol and halothane is significantly potentiated by caffeine and theophylline. The modest uncoupling effects of isoflurane, described here for the first time, are also enhanced by caffeine. Heptanol causes a decrease in [Ca²⁺] _i and [H⁺] _i both in the presence and absence of either caffeine or theophylline. A similar but transient effect on [Ca²⁺] _i is observed with halothane. 4-Aminopyridine strongly inhibits the uncoupling effects of heptanol. The observed decrease in [Ca^2–] _i with heptanol and halothane and negative results obtained with different [Ca²⁺] _o , Ca²⁺-channel blockers (nisoldipine and Cd²⁺) and ryanodine speak against a Ca²⁺ participation. Negative results obtained with 3-isobutyl-l-methylxanthine, forskolin, CPT-cAMP, 8Br-cGMP, adenosine, phorbol ester and H7, superfused in the presence and absence of caffeine and/or heptanol. indicate that neither the heptanol effects nor their potentiation by caffeine are mediated by cyclic nucleotides, adenosine receptors and kinase C. The data suggest a direct effect of anesthetics. possibly involving both polar and hydrophobic interactions with channel proteins. Xanthines and 4-aminopyridine may participate by influencing polar interactions. The potentiating effect of xanthines on cell-to-cell uncoupling by anesthetics may provide some clues on the nature of cardiac arrhythmias in patients treated with theophylline during halothane anesthesia.     

Pleiotropic Effects of Bitter Taste Receptors on [Ca2+]i Mobilization,Hyperpolarization, and Relaxation of Human Airway Smooth Muscle Cells

Asthma is characterized by airway inflammation and airflow obstruction from human airway smooth muscle (HASM) constriction due to increased local bronchoconstrictive substances. We have recently found bitter taste receptors (TAS2Rs) on HASM, which increase [Ca²⁺]_i and relax the muscle. We report here that some, but not all, TAS2R agonists decrease [Ca²⁺]_i and relax HASM contracted by G-protein coupled receptors (GPCRs) that stimulate [Ca²⁺]_i. This suggests both a second pathway by which TAS2Rs relax, and, a heterogeneity of the response phenotype. We utilized eight TAS2R agonists and five procontractile GPCR agonists in cultured HASM cells. We find that heterogeneity in the inhibitory response hinges on which procontractile GPCR is activated. For example, chloroquine inhibits [Ca²⁺]_i increases from histamine, but failed to inhibit [Ca²⁺]_i increases from endothelin-1. Conversely, aristolochic acid inhibited [Ca²⁺]_i increases from endothelin-1 but not histamine. Other dichotomous responses were found when [Ca²⁺]_i was stimulated by bradykinin, angiotensin, and acetylcholine. There was no association between [Ca²⁺]_i inhibition and TAS2R subtype, nor whether [Ca²⁺]_i was increased by G_q- or G_i-coupled GPCRs. Selected studies revealed a correlation between [Ca²⁺]_i inhibition and HASM cell-membrane hyperpolarization. To demonstrate physiologic correlates, ferromagnetic beads were attached to HASM cells and cell stiffness measured by magnetic twisting cytometry. Consistent with the [Ca²⁺]_i inhibition results, chloroquine abolished the cell stiffening response (contraction) evoked by histamine but not by endothelin-1, while aristolochic acid inhibited cell stiffening from endothelin-1, but not from histamine. In studies using intact human bronchi, these same differential responses were found. Those TAS2R agonists that decreased [Ca²⁺]_i, promoted hyperpolarization, and decreased HASM stiffness, caused relaxation of human airways. Thus TAS2Rs relax HASM in two ways: a low-efficiency de novo [Ca²⁺]_i stimulation, and, a high-efficiency inhibition of GPCR-stimulated [Ca²⁺]_i. Furthermore, there is an interaction between TAS2Rs and some GPCRs that facilitates this [Ca²⁺]_i inhibition limb.     

Delayed activation of plasma membrane Ca pump in human neutrophils

The interplay between Ca²⁺ efflux mechanisms of the plasma membrane (PM) and transient changes of the cytosolic concentration of ionized calcium ([Ca²⁺]_i) was studied in suspensions of human neutrophils loaded with the [Ca²⁺]_i indicator, Fura-2. To reveal Ca²⁺ efflux through PM the interference of intracellular Ca stores was prevented by preincubating the cells in the presence of EGTA, thapsigargin, and ionomycin. Addition of econazole prevented varying entry of divalent cations regulated by the filling state of Ca stores. The preincubation seemed to empty and permeabilize virtually all Ca stores, ensuring that the monitored changes of [Ca²⁺]_i were caused exclusively by PM Ca²⁺ transporters. Following preincubation, the addition of CaCl₂ induced, mediated by ionomycin, a transient rise of [Ca²⁺]_i, a spike, eventually decreasing to an intermediary [Ca²⁺]_i level. The ATP-dependent decrease of [Ca²⁺]_i terminating the spike was abolished by the calmodulin antagonist, N-(6-aminohexyl)-1-naphthalenesulfonamide (W-7), but not by the protein kinase C inhibitor, staurosporine, nor by Na⁺-free medium, suggesting that neither activity of protein kinase C nor exchange was necessary for generation of the Ca²⁺ spike. In conclusion, the PM Ca²⁺ pump was responsible for the Ca²⁺ spike by responding to the rapid rise of [Ca²⁺]_i by a delayed activation, possibly involving calmodulin. This characteristic feature of the PM pump may be important for the generation of cellular [Ca²⁺]_i spikes in general.     

Focal increases in [Ca]i may account for apparent low Ca sensitivity in swine carotid artery

Estimates of [Ca²⁺]_i sensitivity in intact smooth muscle are frequently obtained by measuring [Ca²⁺]_i with indicators such as aequorin or Fura-2. We investigated whether focal in increases in [Ca²⁺]_i could impair such measures of [Ca²⁺]_i sensitivity. Stimulation of swine carotid artery with 10 μM histamine increased aequorin estimated [Ca²⁺]_i, Fura-2 estimated [Ca²⁺]_i and Ca²⁺ sensitivity without significantly altering the aequorin/Fura-2 ratio (an estimate of [Ca²⁺]_i homogeneity). Subsequent inhibition of Na⁺/Ca²⁺ exchange by replacement of Na⁺ in the PSS with choline⁺ significantly increased aequorin-estimated [Ca²⁺]_i but only minimally increased Fura-2 estimated [Ca²⁺]_i, myosin light chain (MLC) phosphorylation and force. This resulted in a large increase in the aequorin/Fura-2 ratio, suggesting an increase in [Ca²⁺] inhomogeneity. Addition of 100 μM histamine to tissues in the choline⁺ buffer initially increased both aequorin and Fura-2 estimated [Ca²⁺]_i but after 10 min exposure both of the [Ca²⁺]_i estimates declined to pre-histamine levels. Histamine addition significantly increased MLC phosphorylation and force, indicating increased Ca²⁺ sensitivity, but the aequorin/Fura-2 ratio remained elevated and uncharged from pre-histamine values. These data show that under certain conditions, aequorin and Fura-2 can yield widely differing estimates of [Ca²⁺]_i, and thus can cause misleading assessments of Ca²⁺ sensitization mechanisms. These discrepancies may arise from inhomogeneous or focal increases in [Ca²⁺]_i which can be evaluated with the aequorin/Fura-2 ratio.     

Ratio-imaging of Ca2+i in the self-incompatibility response in pollen tubes of Papaver rhoeas

The data presented here describe ratio-imaging of in intracellular free calcium (Ca²⁺_i) during the self-incompatibility (SI) response in pollen. Use of the ratiometric indicator, fura-2 dextran, in pollen tubes of Papaver rhoeas has provided new, detailed information about the spatial-temporal alterations in Ca²⁺_i, and has permitted calibration of alterations in the concentration of intracellular free calcium ([Ca²⁺]_i) in the SI response. Ratio images demonstrate that, like other pollen tubes, normally growing P. rhoeas pollen tubes exhibit a tip-focused gradient of Ca^2+bf_i, with levels reaching 1–2 μM at the extreme apex of the pollen tube. Non-growing pollen tubes did not exhibit this tip-focused gradient. Basal levels of Ca²⁺_i in the shank of the pollen tube were fairly consistent and had a mean value of 210 nM, with low-level fluctuations +/? 50 nM observed. Challenge with incompatible S proteins resulted in S-specific, rapid and dramatic alterations in [Ca²⁺]_i within a few seconds of challenge. Increases in [Ca²⁺]_i were visualized in the subapical/shank regions of the pollen tube and alterations in [Ca²⁺]_i in this region subsequently increased for several minutes, reaching> 1.5 μM. At the pollen tube tip, a diminution of the tip-focused gradient was observed, which following some fluctuation, was reduced to basal levels within ～1 min. Our data suggest that some of these alterations in [Ca²⁺]_i might be interpreted as a calcium wave, as the changes are not global. Although the increases in [Ca²⁺]_i in the subapical/shank region are very rapid, because tip [Ca²⁺]_i oscillates during normal growth, it is difficult to ascertain whether the increases in the shank of the pollen tube precede the decreases in [Ca²⁺]_i at the pollen tube tip.     

Differential Regulation of Calcium-Activated Potassium Channels by Dynamic Intracellular Calcium Signals

Calcium (Ca²⁺)-activated K⁺ (K_Ca) channels regulate membrane excitability and are activated by an increase in cytosolic Ca²⁺ concentration ([Ca²⁺]_i), leading to membrane hyperpolarization. Most patch clamp experiments that measure K_Ca currents use steady-state [Ca²⁺] buffered within the patch pipette. However, when cells are stimulated physiologically, [Ca²⁺]_i changes dynamically, for example during [Ca²⁺]_i oscillations. Therefore, the aim of the present study was to examine the effect of dynamic changes in [Ca²⁺]_i on small (SK3), intermediate (hIK1), and large conductance (BK) channels. HEK293 cells stably expressing each K_Ca subtype in isolation were used to simultaneously measure agonist-evoked [Ca²⁺]_i signals, using indo-1 fluorescence, and current/voltage, using perforated patch clamp. Agonist-evoked [Ca²⁺]_i oscillations induced a corresponding K_Ca current that faithfully followed the [Ca²⁺]_i in 13–50% of cells, suggesting a good synchronization. However, [Ca²⁺]_i and K_Ca current was much less synchronized in 50–76% of cells that exhibited Ca²⁺-independent current events (55% of SK3-, 50% of hIK1-, and 53% of BK-expressing cells) and current-independent [Ca²⁺]_i events (18% SK3- and 33% of BK-expressing cells). Moreover, in BK-expressing cells, where [Ca²⁺]_i and K_Ca current was least synchronized, 36% of total [Ca²⁺]_i spikes occurred without activating a corresponding K_Ca current spike, suggesting that BK_Ca channels were either inhibited or had become desensitized. This desynchronization between dynamic [Ca²⁺]_i and K_Ca current suggests that this relationship is more complex than could be predicted from steady-state [Ca²⁺]_i and K_Ca current. These phenomena may be important for encoding stimulus–response coupling in various cell types.