Cardiac functions and taurine's actions at different extracellular calcium concentrations in forced swimming stress-loaded rats

Modulation of the sinus rate and contractile force by taurine at different extracellular Ca²⁺ concentrations ([Ca²⁺]_o) was examined using rat right atria loaded with forced swimming stress. Serum concentration of corticosterone profoundly increased in stress-loaded rats as compared with native rats. The taurine level in serum also increased in stress-loaded rats, but was not changed in the different heart tissues and aorta. Heat-shock protein (HSP72) was detectable in cardiac muscles and in the lumen of cardiac blood vessels of stress-loaded rats using a monoclonal antibody. Increasing [Ca²⁺]_o (from 0.9 to 3.6 mM) enhanced the sinus rate and contractile force in a [Ca²⁺]_o-dependent fashion in native rats, but not in stress-loaded rats. Taurine (1–20 mM) caused a negative chronotropic and inotropic effect in a concentration-dependent manner. At 1.8 mM [Ca²⁺]_o, the negative chronotropic effect of taurine (10–20 mM) was attenuated in stress-loaded rats as compared with native rats. These results indicate that swimming stress causes a release of taurine into the serum and reduces the sensitivity to [Ca²⁺]_o. Taurine administration might, in part, exhibit the protective actions on acute stress-induced responses.     

Dependence of the resting [Ca2+]cyt of RBL-1 cells on Ca2+ entry

The cytoplasmic Ca²⁺ concentration ([Ca²⁺]_cyt) in resting cells in an equilibrium between several influx and efflux mechanisms. Here we address the question of whether capacitative Ca²⁺ entry to some extent is active at resting conditions and therefore is part of processes that guarantee a constant [Ca²⁺]_cyt. We measured changes of [Ca²⁺]_cyt in RBL-1 cells with fluorometric techniques. An increase of the extracellular [Ca²⁺] from 1.3 mM to 5 mM induced an incrase in [Ca²⁺]_cyt from 105±10 nM to 145±8.5 nM. This increase could be inhibited by 10 μM Gd³⁺, 10 μM La³⁺ or 50 μM 2-aminoethoxydiphenyl borate, blockers of capacitative Ca²⁺ entry. Application of those blockers to a resting cell in a standard extracellular solution (1.3 mM Ca²⁺) resulted in a decrease of [Ca²⁺]_cyt from 105±10 nM to 88.5±10 nM with La³⁺, from 103±12 to 89±12 nM with Gd³⁺ and from 102±12 nM to 89.5±5 nM with 2-aminoethoxydiphenyl borate. From these data, we conclude that capacitative Ca²⁺ entry beside its function in Ca²⁺ signaling contributes to the regulation of resting [Ca²⁺]_cyt.     

Light-induced extracellular calcium and sodium concentration changes in the retina of Calliphora: involvement in the mechanism of light adaptation

Summary Ion-selective microelectrodes inserted into the compound eyes of Calliphora were used to monitor the changes in extracellular concentration of Ca²⁺ and Na⁺ (Ca_o, Na^o) brought about by a 1-min exposure to white light (maximal luminous intensity 0.1 cd/cm²).Using Ringer solution as the reference (Ca²⁺ = 1 mM), the dark concentration of the calcium in the retina was found to be (1.4 ± 0.4) mM (n=12). Stimulation with light reduces Ca_o. At intensities near maximal the Ca_o signal is phasic, reaching a transient minimum about 6 s after light onset and then rising to a nearly stable plateau below the dark level (-3.3% ± 2.6%). Ca_o signals measured in the white-eyed mutant (chalky), which lacks pigment granules, are comparable to those in the wild type.Conclusions: (a) There are no extracellular Ca²⁺ binding sites that regulate light adaptation, such as were postulated by Hochstrate and Hamdorf (1985). (b) Ca²⁺ influx into the photoreceptors seems to be necessary for light adaptation, (c) The pigment granules have no major function in intracellular calcium regulation.The time course of the Na_o signals resembles that of the Ca_o signals. Because the percentage concentration change is small, light-induced extracellular Na⁺-depletion cannot contribute to a reduced response amplitude at light adaptation.Abbreviations Ca _i intracellular Ca²⁺ concentration - Ca _o extracellular Ca²⁺ concentration - Kino extracellular K⁺ concentration - Na _o extracellular Na⁺ concentration     

Identification of an l-Phenylalanine Binding Site Enhancing the Cooperative Responses of the Calcium-sensing Receptor to Calcium

Functional positive cooperative activation of the extracellular calcium ([Ca²⁺]_o)-sensing receptor (CaSR), a member of the family C G protein-coupled receptors, by [Ca²⁺]_o or amino acids elicits intracellular Ca²⁺ ([Ca²⁺]_i) oscillations. Here, we report the central role of predicted Ca²⁺-binding site 1 within the hinge region of the extracellular domain (ECD) of CaSR and its interaction with other Ca²⁺-binding sites within the ECD in tuning functional positive homotropic cooperativity caused by changes in [Ca²⁺]_o. Next, we identify an adjacent l-Phe-binding pocket that is responsible for positive heterotropic cooperativity between [Ca²⁺]_o and l-Phe in eliciting CaSR-mediated [Ca²⁺]_i oscillations. The heterocommunication between Ca²⁺ and an amino acid globally enhances functional positive homotropic cooperative activation of CaSR in response to [Ca²⁺]_o signaling by positively impacting multiple [Ca²⁺]_o-binding sites within the ECD. Elucidation of the underlying mechanism provides important insights into the longstanding question of how the receptor transduces signals initiated by [Ca²⁺]_o and amino acids into intracellular signaling events.     

Extracellular Calcium Sensing by Glial Cells: Low Extracellular Calcium Induces Intracellular Calcium Release and Intercellular Signaling

Abstract: Glial cells in primary mixed cultures or purified astrocyte cultures from mouse cortex respond to reduced extracellular calcium concentration ([Ca²⁺]_e) with increases in intracellular calcium concentration ([Ca²⁺]_i) that include single-cell Ca²⁺ oscillations and propagated intercellular Ca²⁺ waves. The rate and pattern of propagation of low [Ca²⁺]_e-induced intercellular Ca²⁺ waves are altered by rapid perfusion of the extracellular medium, suggesting the involvement of an extracellular messenger in Ca²⁺ wave propagation. The low [Ca²⁺]_e-induced Ca²⁺ response is abolished by thapsigargin and by the phospholipase antagonist U73122. The low [Ca²⁺]_e-induced response is also blocked by replacement of extracellular Ca²⁺ with Ba²⁺, Zn²⁺, or Ni²⁺, and by 100 µM La³⁺. Glial cells in lowered [Ca²⁺]_e(0.1–0.5 mM) show an increased [Ca²⁺]_i response to bath application of ATP, whereas glial cells in increased [Ca²⁺]_e (10–15 mM) show a decreased [Ca²⁺]_i response to ATP. These results show that glial cells possess a mechanism for coupling between [Ca²⁺]_e and the release of Ca²⁺ from intracellular stores. This mechanism may be involved in glial responses to the extracellular environment and may be important in pathological conditions associated with low extracellular Ca²⁺ such as seizures or ischemia.     

Suppression of programmed neuronal death by a thapsigargin-induced Ca2+ influx

Rat sympathetic neurons undergo programmed cell death (PCD) in vitro and in vivo when they are deprived of nerve growth factor (NGF). Chronic depolarization of these neurons in cell culture with elevated concentrations of extracellular potassium ([K⁺]_o) prevents this death. The effect of prolonged depolarization on neuronal survival is thought to be mediated by a rise of intracellular calcium concentration ([Ca²⁺]_i) caused by Ca²⁺ influx through voltage-gated channels. In this report we investigate the effects of chronic treatment of rat sympathetic neurons with thapsigargin, an inhibitor of intracellular Ca²⁺ sequestration. In medium containing a normal concentration of extracellular Ca²⁺ ([Ca²⁺]_o), thapsigargin caused a sustained rise of intracellular Ca²⁺ concentration and partially blocked death of NGF-deprived cells. Elevating [Ca²⁺]_o in the presence of thapsigargin further increased [Ca²⁺]_i, suggesting that the sustained rise of [Ca²⁺]_i was caused by a thapsigargin-induced Ca²⁺ influx. This treatment potentiated the effect of thapsigargin on survival. The dihydropyridine Ca²⁺ channel antagonist, nifedipine, blocked both a sustained elevation of [Ca²⁺]_i and enhanced survival caused by depolarization with elevated [K⁺]_o, suggesting that these effects are mediated by Ca²⁺ influx through L-type channels. Nifedipine did not block the sustained rise of [Ca²⁺]_i or enhanced survival caused by thapsigargin treatment, indicating that these effects were not mediated by influx of Ca²⁺ through L-type channels. These results provide additional evidence that increased [Ca²⁺]_i can suppress neuronal PCD and identify a novel method for chronically raising neuronal [Ca²⁺]_i for investigation of this and other Ca²⁺-dependent phenomena. © 1995 John Wiley & Sons, Inc.     

Elevation of Extracellular Ca2+ Induces Store-Operated Calcium Entry via Calcium-Sensing Receptors: A Pathway Contributes to the Proliferation of Osteoblasts

Aims

The local concentration of extracellular Ca²⁺ ([Ca²⁺]_o) in bone microenvironment is accumulated during bone remodeling. In the present study we investigated whether elevating [Ca²⁺]_o induced store-operated calcium entry (SOCE) in primary rat calvarial osteoblasts and further examined the contribution of elevating [Ca²⁺]_o to osteoblastic proliferation.

Methods

Cytosolic Ca²⁺ concentration ([Ca²⁺]_c) of primary cultured rat osteoblasts was detected by fluorescence imaging using calcium-sensitive probe fura-2/AM. Osteoblastic proliferation was estimated by cell counting, MTS assay and ATP assay. Agonists and antagonists of calcium-sensing receptors (CaSR) as well as inhibitors of phospholipase C (PLC), SOCE and voltage-gated calcium (Cav) channels were applied to study the mechanism in detail.

Results

Our data showed that elevating [Ca²⁺]_o evoked a sustained increase of [Ca²⁺]_c in a dose-dependent manner. This [Ca²⁺]_c increase was blocked by TMB-8 (Ca²⁺ release inhibitor), 2-APB and BTP-2 (both SOCE blockers), respectively, whereas not affected by Cav channels blockers nifedipine and verapamil. Furthermore, NPS2143 (a CaSR antagonist) or {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 (a PLC inhibitor) strongly reduced the [Ca²⁺]_o-induced [Ca²⁺]_c increase. The similar responses were observed when cells were stimulated with CaSR agonist spermine. These data indicated that elevating [Ca²⁺]_o resulted in SOCE depending on the activation of CaSR and PLC in osteoblasts. In addition, high [Ca²⁺]_o significantly promoted osteoblastic proliferation, which was notably reversed by BAPTA-AM (an intracellular calcium chelator), 2-APB, BTP-2, TMB-8, NPS2143 and {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122, respectively, but not affected by Cav channels antagonists.

Conclusions

Elevating [Ca²⁺]_o induced SOCE by triggering the activation of CaSR and PLC. This process was involved in osteoblastic proliferation induced by high level of extracellular Ca²⁺ concentration.     

A Prototypic Intracellular Calcium Antagonist, TMB-8, Protects Cultured Cerebellar Granule Cells Against the Delayed, Calcium-Dependent Component of Glutamate Neurotoxicity

Abstract: The effect(s) of a prototypic intracellular Ca²⁺ antagonist, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), on glutamate-induced neurotoxicity was investigated in primary cultures of mouse cerebellar granule cells. Glutamate evoked an increase in cytosolic free-Ca²⁺ levels ([Ca²⁺]_i) that was dependent on the extracellular concentration of Ca²⁺ ([Ca²⁺]_o). In addition, this increase in [Ca²⁺]_i correlated with a decrease in cell viability that was also dependent on [Ca²⁺]_o. Glutamate-induced toxicity, quantified by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) staining, was shown to comprise two distinct components, an “early” Na⁺/Cl^?-dependent component observed within minutes of glutamate exposure, and a “delayed” Ca²⁺-dependent component (ED₅₀～50 µM) that coincided with progressive degeneration of granule cells 4–24 h after a brief (5–15 min) exposure to 100 µM glutamate. Quantitative analysis of cell viability and morphological observations identify a “window” in which TMB-8 (at >100 µM) protects granule cells from the Ca²⁺-dependent, but not the Na⁺/Cl^?-dependent, component of glutamate-induced neurotoxic damage, and furthermore, where TMB-8 inhibits glutamate-evoked increases in [Ca²⁺]_i. These findings suggest that Ca²⁺ release from a TMB-8-sensitive intracellular store may be a necessary step in the onset of glutamate-induced excitotoxicity in granule cells. However, these conclusions are compromised by additional observations that show that TMB-8 (1) exhibits intrinsic toxicity and (2) is able to reverse its initial inhibitory action on glutamate-evoked increases in [Ca²⁺]_i and subsequently effect a pronounced time-dependent potentiation of glutamate responses. Dantrolene, another putative intracellular Ca²⁺ antagonist, was completely without effect in this system with regard to both glutamate-evoked increases in [Ca²⁺]_i and glutamate-induced neurotoxicity.     

Involvement of protein kinase C-alpha and -epsilon in extracellular Ca signalling mediated by the calcium sensing receptor

The sensing of extracellular Ca²⁺ concentration ([Ca²⁺]_o) and modulation of cellular processes associated with acute or sustained changes in [Ca²⁺]_o are cell-type specific and mediated by the calcium sensing receptor (CaR). [Ca²⁺]_o signalling requires protein kinase C (PKC), but the identity and role of PKC isoforms in CaR-mediated responses remain unclear. Here we show that high [Ca²⁺]_o activated PKC-α and PKC-ε in parathyroid cells and in human embryonic kidney (HEK293) cells overexpressing the CaR (HEK-CaR) and that this response correlated with the CaR-dependent activation of mitogen-activated protein kinases ERK1/2. Activation of ERK1/2 by acute high [Ca²⁺]_o required influx of Ca²⁺through Ni²⁺-sensitive Ca²⁺channels and phosphatidylinositol-dependent phospholipase C-β activity. Inhibition of PKC by co-expression of dominant-negative (DN) mutants of PKC-α or -ε with the CaR attenuated sustained ERK1/2 activation. Overexpression of a PKC phosphorylation site (T888A) mutant CaR in HEK293 cells showed that this site was important for ERK1/2 activation at high [Ca²⁺]_o. Activation of ERK1/2 by high [Ca²⁺]_o was not necessary for the [Ca²⁺]_o-regulated secretion of parathyroid hormone (PTH) in dispersed bovine parathyroid cells. These data suggest that the CaR-mediated [Ca²⁺]_o signal leading to regulated PTH secretion that requires diacylglycerol-responsive PKC isoforms is not mediated via the ERK pathway.     

Calcium mobilization and muscle contraction induced by acetylcholine in swine trachealis

The roles of Ca²⁺ mobilization in development of tension induced by acetylcholine (ACh, 0.1–100 µM) in swine tracheal smooth muscle strips were studied. Under control conditions, ACh induced a transient increase in free cytosolic calcium concentration ([Ca²⁺]_i) that declined to a steady-state level. The peak increase in [Ca²⁺]_i correlated with the magnitude of tension at each [ACh] after a single exposure to ACh, while the steady-state [Ca²⁺]_i did not. Removal of extracellular Ca²⁺ had little effect on peak [Ca²⁺]_i but greatly reduced steady-state increases in [Ca²⁺]_i and tension. Verapamil inhibited steady-state [Ca²⁺]_i only at [ACh]<1 µM. After depletion of internal Ca²⁺ stores by 10 min exposure to ACh in Ca²⁺-free solution and then washout of ACh for 5 min in Ca²⁺-free solution, simultaneous re-exposure to ACh in the presence of 2.5 mM Ca²⁺ increased [Ca²⁺]_i to the control steady-state level without overshoot. The tension attained was the same as control for each [ACh] used. Continuous exposure to successively increasing [ACh] (0.1–100 µM) also reduced the overshoot of [Ca²⁺]_i at 10 and 100 µM ACh, yet tension reached control levels at each [ACh] used. We conclude that the steady-state increase in [Ca²⁺]_i is necessary for tension maintenance and is dependent on Ca²⁺ influx through voltage-gated calcium channels at 0.1 µM ACh and through a verapamil-insensitive pathway at 10 and 100 µM. The initial transient increase in calcium arises from intracellular stores and is correlated with the magnitude of tension only in muscles that have completely recovered from previous exposure to agonists.     

Osmotic Swelling-Induced Changes in Cytosolic Calcium Do Not Affect Regulatory Volume Decrease in Rat Cultured Suspended Cerebellar Astrocytes

Abstract: Hyposmotic swelling-induced changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i) and their influence on regulatory volume decrease (RVD) were examined in rat cultured suspended cerebellar astrocytes. Hyposmotic media (50 or 30%) evoked an immediate rise in [Ca²⁺]_i from 117 nM to a mean peak increase of 386 (50%) and 220 nM (30%), followed by a maintained plateau phase. Ca²⁺ influx through the plasmalemma as well as release from internal stores contributed to this osmosensitive [Ca²⁺]_i elevation. Omission of external Ca²⁺ or addition of Cd²⁺, Mn²⁺, or Gd³⁺ did not reduce RVD, although it was decreased by La³⁺ (0.1–1 mM). Verapamil did not affect either the swelling-evoked [Ca²⁺]_i or RVD. Maneuvers that deplete endoplasmic reticulum (ER) Ca²⁺ stores, such as treatment (in Ca²⁺-free medium) with 0.2 µM thapsigargin (Tg), 10 µM 2,5-di-tert-butylhydroquinone, 1 µM ionomycin, or 100 µM ATP abolished the increase in [Ca²⁺]_i but did not affect RVD. However, prolonged exposure to 1 µM Tg blocked RVD regardless of ER Ca²⁺ content or cytosolic Ca²⁺ levels. Ryanodine (up to 100 µM) and caffeine (10 mM) did not modify [Ca²⁺]_i or RVD. BAPTA-acetoxymethyl ester (20 µM) abolished [Ca²⁺]_i elevation without affecting RVD, but at higher concentrations BAPTA prevented cell swelling and blocked RVD. We conclude that the osmosensitive [Ca²⁺]_i rise occurs as a consequence of increased Ca²⁺ permeability of plasma and organelle membranes, but it appears not relevant as a transduction signal for RVD in rat cultured cerebellar astrocytes.     

Methylmercury-Induced Elevations in Intrasynaptosomal Zinc Concentrations: An 19F-NMR Study

Abstract: Methylmercury (MeHg) increases the concentration of intracellular Ca²⁺ ([Ca²⁺]_i) and another endogenous polyvalent cation in both synaptosomes and NG108-15 cells. In synaptosomes, the elevation in [Ca²⁺]_i was strictly dependent on extracellular Ca²⁺ (Ca²⁺_e); similarly, in NG108-15 cells, a component of the elevations in [Ca²⁺]_i was Ca²⁺_e dependent. The MeHg-induced elevations in endogenous polyvalent cation concentration were independent of Ca²⁺_e in synaptosomes and NG108-15 cells. The pattern of alterations in fura-2 fluorescence suggested the endogenous polyvalent cation may be Zn²⁺. Using ¹⁹F-NMR spectroscopy of rat cortical synaptosomes loaded with the fluorinated chelator 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N′,N′-tetraacetic acid (5F-BAPTA), we have determined unambiguously that MeHg increases the free intrasynaptosomal Zn²⁺ concentration ([Zn²⁺]_i). In buffer containing 200 µM EGTA to prevent the Ca²⁺_e-dependent elevations in [Ca²⁺]_i, the [Zn²⁺]_i was 1.37 ± 0.20 nM; following a 40-min exposure to MeHg-free buffer [Zn²⁺]_i was 1.88 ± 0.53 nM. Treatment of synaptosomes for 40 min with 125 µM MeHg yielded [Zn²⁺]_i of 2.69 ± 0.55 nM, whereas 250 µM MeHg significantly elevated [Zn²⁺]_i to 3.99 ± 0.68 nM. No Zn²⁺ peak was observed in synaptosomes treated with the cell-permeant heavy metal chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN, 100 µM) following 250 µM MeHg exposure. [Ca²⁺]_i in buffer containing 200 µM EGTA was 338 ± 26 nM and was 370 ± 64 nM following an additional 40-min exposure to MeHg-free buffer. [Ca²⁺]_i was 498 ± 28 or 492 ± 53 nM during a 40-min exposure to 125 or 250 µM MeHg, respectively. None of the values of [Ca²⁺]_i differed significantly from either pretreatment levels or buffer-treated controls.     

The role of magnesium in regulating CCK-8-evoked secretory responses in the exocrine rat pancreas

This study investigates the effect of magnesium (Mg²⁺) on the secretory responses and the mobilization of calcium (Ca²⁺) and Mg²⁺ evoked by cholecystokinin-octapeptide (CCK-8) in the exocrine rat pancreas. In the isolated intact perfused pancreas CCK-8 (10^–10 M) produced marked increases in juice flow and total protein output in zero and normal (1.1 mM) extracellular Mg²⁺ [Mg²⁺]_o compared to a much reduced secretory response in elevated (5 mM and 10 mM) [Mg²⁺]_o Similar effects of perturbation of [Mg²⁺]_o on amylase secretion and ⁴⁵Ca²⁺ uptake (influx) were obtained in isolated pancreatic segments. In pancreatic acinar cells loaded with the fluorescent bioprobe fura-2 acetomethylester (AM), CCK-8 evoked marked increases in cytosolic free Ca²⁺ concentration [Ca²⁺]_i in zero and normal [Mg²⁺]_o compared to a much reduced response in elevated [Mg²⁺]_o Pretreatment of acinar cells with either dibutyryl cyclic AMP (DB₂ cAMP) or forskolin had no effect on the CCK-8 induced changes in [Ca²⁺]_i. In magfura-2-loaded acinar cells CCK-8 (10^–8 M) stimulated an initial transient rise in intracellular free Mg²⁺ concentration [Mg²⁺]_i followed by a more prolonged and sustained decrease. This response was abolished when sodium Na⁺ was replaced with N-methyl-D-glucamine (NMDG). Incubation of acinar cells with 10 mM Mg²⁺ resulted in an elevation in [Mg²⁺]_i. Upon stimulation with CCK-8, [Mg²⁺]_i. decreased only slightly compared with the response obtained in normal [Mg²⁺]_o. CCK-8 caused a net efflux of Mg²⁺ in pancreatic segments; this effect was abolished when extracellular sodium [Na⁺]_o was replaced with either NMDG or choline. The results indicate that Mg²⁺ can regulate CCK-8-evoked secretory responses in the exocrine pancreas possibly via Ca²⁺ mobilization. Moreover, the movement of Mg²⁺ in pancreatic acinar cells is dependent upon extracellular Na⁺.     

Activation of Ca2+/Calmodulin-Dependent Protein Kinase II by Extracellular Calcium in Cultured Hippocampal Pyramidal Neurons

Abstract: The ability of various stimuli to convert Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) into a Ca²⁺-independent (autonomous) form was examined in cultured embryonic rat hippocampal pyramidal neurons. The most effective stimulation by far was observed when cells were equilibrated in buffer containing low extracellular [Ca²⁺] ([Ca²⁺]_o) (～50 nM) and then shifted to normal [Ca²⁺]_o (～1.26 mM) by addition of CaCl₂ (referred to as “Ca²⁺ stimulation”). Virtually complete (>90%) conversion of the kinase to the autonomous form occurred within 30–50 s, with a return to baseline within 5 min. By contrast, depolarization of cells with high [K⁺] or treatment with glutamate or a Ca²⁺ ionophore caused insignificant increases (<10%) in levels of the autonomous form. The Ca²⁺-stimulated increase in CaMKII autonomy coincided with a two- to threefold increase in kinase subunit phosphorylation. In the first 40 s of Ca²⁺ stimulation, ³²P incorporation into the immunoprecipitated subunits of CaMKII occurred exclusively on threonine residues, including Thr²⁸⁶Thr²⁸⁷ of the α/β subunits. Longer incubation of cells resulted in a decline of phosphothreonine content, whereas levels of phosphoserine-containing peptides showed a significant increase. The activation of CaMKII by Ca²⁺ stimulation was accompanied by only a small rise in intracellular [Ca²⁺]. Inhibitor studies showed that Na⁺-dependent action potentials and Ca²⁺ influx through glutamate receptors or voltage-sensitive Ca²⁺ channels did not contribute to the activation. Moreover, CaMKII was not activated by extracellular addition of other cations, including Mn²⁺, Mg²⁺, Co²⁺, or Gd³⁺. Although the mechanism of Ca²⁺ stimulation is presently unclear, it may involve either activation of extracellular calcium receptors or capacitative calcium entry. The dramatic rise in CaMKII autonomy and the Ca²⁺ selectivity of the response suggest a direct and specific relationship between [Ca²⁺]_o and the state of activation of the kinase in intact neurons.     

Involvement of Intracellular or Extracellular Calcium in Activation of Tyrosine Hydroxylase Gene Expression in PC12 Cells

Abstract: The relationship between elevations in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) by different mechanisms and tyrosine hydroxylase (TH) gene expression was examined. Depolarization by an elevated K⁺ concentration triggered rapid and sustained increases in [Ca²⁺]_i from a basal level of ～50 to 110–150 nM and three- to fourfold elevations in TH mRNA levels, requiring extracellular calcium but not inositol 1,4,5-trisphosphate (IP₃). On the other hand, bradykinin or thapsigargin, both of which induce release of intracellular calcium stores via IP₃ or inhibition of Ca²⁺-ATPase, rapidly elevated [Ca²⁺]_i to >200 nM and increased TH gene expression (three-to fivefold). Confocal imaging showed that the elevations in [Ca²⁺]_i in each case occurred throughout the cyto- and nucleoplasm. The initial rise in [Ca²⁺]_i due to either bradykinin or thapsigargin, which did not require extracellular calcium, was sufficient to initiate the events leading to increased TH expression. Consistent with this, the effects of bradykinin on TH expression were inhibited by 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid or 3,4,5-trimethoxybenzoic acid 8-(diethylamino)-octyl ester which chelates or inhibits the release of intracellular calcium, respectively. Bradykinin required a rise in [Ca²⁺]_i for <10 min, as opposed to 10–30 min for depolarization to increase TH mRNA levels. These results demonstrate that although each of these treatments increased TH gene expression by raising [Ca²⁺]_i, there are important differences among them in terms of the magnitude of elevated [Ca²⁺]_i, requirements for extracellular calcium or release of intracellular calcium stores, and duration of elevated [Ca²⁺]_i, indicating the involvement of different calcium signaling pathways leading to regulation of TH gene expression.     

Uncoupling secretion and tip growth in lily pollen tubes: evidence for the role of calcium in exocytosis

Cytoplasmic calcium concentration ([Ca²⁺]_i) and extracellular calcium (Ca²⁺_o) influx has been studied in pollen tubes of Lilium longliflorum in which the processes of cell elongation and exocytosis have been uncoupled by use of Yariv phenylglycoside ((β-D-Glc)₃). Growing pollen tubes were pressure injected with the ratio dye fura-2 dextran and imaged after application of (β-D-Glc)₃, which binds arabinogalactan proteins (AGPs). Application of (β-D-Glc)₃ inhibited growth but not secretion. Ratiometric imaging of [Ca²⁺]_i revealed an initial spread in the locus of the apical [Ca²⁺]_i gradient and substantial elevations in basal [Ca²⁺]_i followed by the establishment of new regions of elevated [Ca²⁺]_i on the flanks of the tip region. Areas of elevated [Ca²⁺]_i corresponded to sites of pronounced exocytosis, as evidenced by the formation of wall ingrowths adjacent to the plasma membrane. Ca²⁺_o influx at the tip of (β-D-Glc)₃-treated pollen tubes was not significantly different to that of control tubes. Taken together these data indicate that regions of elevated [Ca²⁺]_i, probably resulting from Ca²⁺_o influx across the plasma membrane, stimulate exocytosis in pollen tubes independent of cell elongation.     

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.     

Regulation of Ion Fluxes,Cell Volume and Gap Junctional Coupling by cGMP in GFSHR-17 Granulosa Cells

Gap junctional communication between granulosa cells seems to play a crucial role for follicular growth and atresia. Application of the double whole-cell patch-clamp- and ratiometric fura-2-techniques allowed a simultaneous measurement of gap junctional conductance (G _j) and cytoplasmic concentration of free Ca²⁺ ([Ca²⁺]_i) in a rat granulosa cell line GFSHR-17. The voltage-dependent gating of G _j varied for different cell pairs. One population exhibited a bell-shape dependence of G _j on transjunctional voltage, which was strikingly similar to that of Cx43/Cx43 homotypic gap junction channels expressed in pairs of oocytes of Xenopus laevis. Within 15–20 min, gap junctional uncoupling occurred spontaneously, which was preceded by a sustained increase of [Ca²⁺]_i and accompanied by shrinkage of cellular volume. These responses to the whole-cell configuration were avoided by absence of extracellular Ca²⁺, blockage of K⁺ efflux, or addition of 8-bromoguanosine 3,5-cyclic monophosphate (8-Br-cGMP) to the pipette solution. Even in the absence of extracellular Ca²⁺ or blockage of K⁺ efflux, formation of whole-cell configuration generated a Ca²⁺ spike that could be suppressed by the presence of 8-Br-cGMP. We propose that intracellular cGMP regulates Ca²⁺ release from intracellular Ca²⁺ stores, which activates sustained Ca²⁺ influx, K⁺ efflux and cellular shrinkage. We discuss whether gap junctional conductance is directly affected by cGMP or by cellular shrinkage and whether gap junctional coupling and/or cell shrinkage is involved in the regulation of apoptotic/necrotic processes in granulosa cells.     

Blockade of insulin sensitive steady-state R-type Ca2+ channel by PN 200-110 in heart and vascular smooth muscle

The effect of high K^– concentration, insulin and the L-type Ca^2– channel blocker PN 200-110 on cytosolic intracellular free calcium ([Ca²⁺]_i) was studied in single ventricular myocytes of 10-day-old embryonic chick heart, 20-week-old human fetus and rabbit aorta (VSM) single cells using the Ca²⁺-sensitive fluorescent dye, Fura-2 microfluorometry and digital imaging technique. Depolarization of the cell membrane of both heart and VSM cells with continuous superfusion of 30 mM [K⁺]_o induced a rapid transient increase of [Ca²⁺]_i that was followed by a sustained component. The early transient increase of [Ca²⁺]_i by high [⁺]_o was blocked by the L-type calcium channel antagonist nifedipine. However, the sustained component was found to be insensitive to this drug. PN 200-110 another L-type Ca²⁺ blocker was found to decrease both the early transient and the sustained increase of [Ca²⁺]_i induced by depolarization of the cell membrane with high [K⁺]_o. Insulin at a concentration of 40 to 80 U/ml only produced a sustained increase of [Ca²⁺]_i that was blocked by PN 200-110 or by lowering the extracellular Ca²⁺ concentration with EGTA. The sustained increase of [Ca²⁺], induced by high [K⁺]_o or insulin was insensitive to metabolic inhibitors such as KCN and ouabain as well to the fast Na⁺ channel blocker, tetrodotoxin and to the increase of intracellular concentrations of cyclic nucleotides. Using the patch clamp technique, insulin did not affect the L-type Ca²⁺ current and the delayed outward K⁺ current. These results suggest that the early increase of (Ca²⁺]_i during depolarization of the cell membrane of heart and VSM cells with high [K⁺]_o is due to the opening and decay of an L-type Ca ²⁺ channel. However, the sustained increase of [Ca²⁺]_i during a sustained depolarization is due to the activation of a resting (R) Ca ²⁺ channel that is insensitive to lowering [ATP]_i and sensitive to insulin.     

Interaction between thapsigargin and ATP4− in the regulation of the intracellular calcium in rat submandibular glands

Rat submandibular glands were digested with crude collagenase, and the intracellular calcium concentration of the cellular suspension was measured using fura-2. In the absence of extracellular magnesium and calcium ([Ca²⁺]_o), ATP had no effect; the response to ATP peaked at 1–2.5 mM [Ca²⁺]_o and was inhibited at 5 mM. One millimolar (mM) extracellular ATP did not increase the leak of LDH or fura-2; 10 m?M Coomassie brilliant blue G specifically inhibited the effect of ATP on [Ca²⁺]_in. Depleting intracellular calcium pools with thapsigargin did not affect the response to ATP. Using a Ca²⁺-free/Ca²⁺ reintroduction protocol, it was shown that ATP and thapsigargin increase the uptake of extracellular calcium. The effect of the two agonists was synergistic. Removal of extracellular sodium inhibited the effect of carbachol on [Ca²⁺]_in and the calcium uptake but potentiated the response to ATP. These results suggest that, after binding to purinergic receptors, extracellular ATP^4- increases [Ca²⁺]_in. ATP^4- does not mobilize thapsigargin-sensitive intracellular calcium pools (among which is the IP₃-sensitive calcium pool) but stimulates the uptake of extracellular calcium by a mechanism inhibited by extracellular sodium, probably by opening a nonselective cation channel. © 1994 Wiley-Liss, Inc.