Calcium signaling: up, down, up, down...what's the point?
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Function associated molecules on nonspecific cytotoxic cells: role in calcium signaling, redirected lysis, and modulation of cytotoxicity.
Monoclonal antibodies (mAbs) specific for a putative antigen receptor (function associated molecule, FAM) on fish NCC were tested for their ability to initiate signal transduction responses. Anti-FAM hybridoma target cells were significantly lysed by NCC when tested by redirected lysis (RL). Redirected lysis was enhanced by 1-h pretreatment of the NCC with anti-FAM mAb; however, 18-h pretreatment produced significant inhibition. The effects of the calcium ionophore A23187 on RL were also examined: 10(-4) M but not 10(-5) M A23187 significantly increased RL. Purified NCC were treated with mAb and cellular DNA synthesis was determined. Significant increases in NCC incorporation of tritiated thymidine were measured 72 h after treatment with mAb. Experiments were also conducted to determine levels of free cytosolic calcium in NCC following mAb binding. Anti-FAM mAb binding produced a rapid rise in cytosolic calcium (200-500 nM) determined by Fura-2/AM fluorescence. Calcium ionophore A23187, PHA, and Con-A treatment of NCC also produced significantly increased [Ca2+]i. Target cell binding by NCC caused a three-fold increase in [Ca2+]i. These data suggest that triggering of the FAM may initiate a signal(s) that activates cytotoxicity, increases cytosolic free calcium concentrations, and initiates DNA synthesis.
Animals↗
Electromagnetic field effects on cells of the immune system: the role of calcium signaling.
During the past decade considerable evidence has accumulated demonstrating that nonthermal exposures of cells of the immune system to extremely low-frequency (ELF) electromagnetic fields (< 300 Hz) can elicit cellular changes that might be relevant to in vivo immune activity. A similar responsiveness to nonionizing electromagnetic energy in this frequency range has also been documented for tissues of the neuroendocrine and musculoskeletal system. However, knowledge about the underlying biological mechanisms by which such fields can induce cellular changes is still very limited. It is generally believed that the cell membrane and Ca(2+)-regulated activity is involved in bioactive ELF field coupling to living systems. This article begins with a short review of the current state of knowledge concerning the effects of nonthermal levels of ELF electromagnetic fields on the biochemistry and activity of immune cells and then closely examines new results that suggest a role for Ca2+ in the induction of these cellular field effects. Based on these findings it is proposed that membrane-mediated Ca2+ signaling processes are involved in the mediation of field effects on the immune system.
Animals↗
Opiate-mediated inhibition of calcium signaling is decreased in dorsal root ganglion neurons from the diabetic BB/W rat.
The effect of diabetes mellitus on opiate-mediated inhibition of calcium current density (I(D Ca) [pA pF-1]) and cytosolic calcium response ([Ca2+]i nM) to depolarization with elevated KCl and capsaicin was assessed. Experiments were performed on isolated, acutely dissociated dorsal root ganglion (DRG) neurons from diabetic, BioBreeding/Worcester (BB/W) rats and age-matched control animals. Sciatic nerve conduction velocity was significantly decreased in diabetic animals compared to controls. Mean I(DCa) and [Ca2+]i responses to capsaicin and elevated KCl recorded in DRGs from diabetic animals were significantly larger than those recorded in DRG neurons from controls. In neurons from diabetic animals, the opiate agonist dynorphin A (Dyn A; 1, 3, and 5 microM) had significantly less inhibitory effect on I(D Ca) and KCl-induced [Ca2+]i responses compared to controls. Omega-conotoxin GVIA (omega-CgTX; 10 microM) and pertussis toxin (PTX; 250 ng ml-1) abolished Dyn A-mediated inhibition of I(DCa) and [Ca2+]i in control and diabetic neurons, suggesting that Dyn A modulated predominantly N-type calcium channels coupled to opiate receptors via PTX-sensitive (Gi/o) inhibitory G proteins. These results suggest that opiate-mediated regulation of PTX-sensitive, G protein-coupled calcium channels is diminished in diabetes and that this correlates with impaired regulation of cytosolic calcium.
Animals↗
Mechanisms of intercellular calcium signaling in glial cells studied with dantrolene and thapsigargin.
Mechanical stimulation of a single cell in a primary mixed glial cell culture induced a wave of increased intracellular calcium concentration ([Ca2+]i) that was communicated to surrounding cells. Following propagation of the Ca2+ wave, many cells showed asynchronous oscillations in [Ca2+]i. Dantrolene sodium (10 microM) inhibited the increase in [Ca2+]i associated with this Ca2+ wave by 60-80%, and prevented subsequent Ca2+ oscillations. Despite the markedly decreased magnitude of the increase in [Ca2+]i, the rate of propagation and the extent of communication of the Ca2+ wave were similar to those prior to the addition of dantrolene. Thapsigargin (10 nM to 1 microM) induced an initial increase in [Ca2+]i ranging from 100 nM to 500 nM in all cells that was followed by a recovery of [Ca2+]i to near resting levels in most cells. Transient exposure to thapsigargin for 2 min irreversibly blocked communication of Ca2+ wave from the stimulated cell to adjacent cells. Glutamate (50 microM) induced an initial increase in [Ca2+]i in most cells that was followed by sustained oscillations in [Ca2+]i in some cells. Dantrolene (10 microM) inhibited this initial [Ca2+]i increase caused by glutamate by 65-90% and abolished subsequent oscillations. Thapsigargin (10 nM to 1 micron) abolished the response to glutamate in over 99% of cells. These results suggest that while both dantrolene and thapsigargin inhibit intracellular Ca2+ release, only thapsigargin affects the mechanism that mediates intercellular communication of Ca2+ waves. These findings are consistent with the hypothesis that inositol trisphosphate (IP3) mediates the propagation of Ca2+ waves whereas Ca(2+)-induced Ca2+ release amplifies Ca2+ waves and generates subsequent Ca2+ oscillations.
Animals↗
Intercellular calcium signaling via gap junction in connexin-43-transfected cells.
In excitable cells, intracellular Ca2+ is released via the ryanodine receptor from the intracellular Ca2+ storing structure, the sarcoplasmic reticulum. To determine whether this released Ca2+ propagates through gap junctions to neighboring cells and thereby constitutes a long range signaling network, we developed a cell system in which cells expressing both connexin-43 and ryanodine receptor are surrounded by cells expressing only connexin-43. When the ryanodine receptor in cells was activated by caffeine, propagation of Ca2+ from these caffeine-responsive cells to neighboring cells was observed with a Ca2+ imaging system using fura-2/AM. Inhibitors of gap junctional communication rapidly and reversibly abolished this propagation of Ca2+. Together with the electrophysiological analysis of transfected cells, the observed intercellular Ca2+ wave was revealed to be due to the reconstituted gap junction of transfected cells. We next evaluated the functional roles of cysteine residues in the extracellular loops of connexin-43 in gap junctional communication. Mutations of Cys54, Cys187, Cys192, and Cys198 to Ser showed the failure of Ca2+ propagation to neighboring cells in accordance with the electrical uncoupling between transfected cells, whereas mutations of Cys61 and Cys68 to Ser showed the same pattern as the wild type. [14C]Iodoacetamide labeling of free thiols of cysteine residues in mutant connexin-43s showed that two pairs of intramolecular disulfide bonds are formed between Cys54 and Cys192 and between Cys187 and Cys198. These results suggest that intercellular Ca2+ signaling takes place in cultured cells expressing connexin-43, leading to their own synchronization and that the extracellular disulfide bonds of connexin-43 are crucial for this process.
Calcium↗
Intracellular calcium signalling after binding of low-density lipoprotein to confluent and nonconfluent cultures of an endothelial cell line, EA.hy 926.
The effect of lipoproteins on cellular calcium ion concentration ([Ca2+]i) in EA.hy 926 endothelial cells was investigated, particularly with respect to the difference in response on [Ca2+]i between native low-density lipoprotein (LDL) which binds to the apo B/E receptor, and acetylated LDL (AcLDL) which binds to the scavenger receptor. The scavenger receptor recognizes chemically or cell-induced modified LDL. LDL as well as AcLDL caused a transient increase of [Ca2+]i lasting 1-2 min. On a protein basis, LDL was more effective that AcLDL in raising [Ca2+]i. Preincubation of confluent cultures in growth medium with a reduced fetal bovine serum content (2% FBS instead of 10%) increased the potency of LDL to increase [Ca2+]i. The LDL-induced peak [Ca2+]i was dependent on cell density. The effect of AcLDL on [Ca2+]i did not differ between confluent and nonconfluent cultures. Also, preincubation with 2% FBS did not modify the AcLDL-induced calcium response. We conclude that binding of lipoproteins to membrane lipoprotein receptors is responsible for the transient rise of [Ca2+]i although the characteristics of the calcium response are dependent on the receptor involved, i.e. the apo B/E (LDL) receptor or the scavenger receptor. We suggest that Ca2+ acts as a second messenger, and that the LDL-induced calcium response is controlled by the proliferative state of the cells.
Calcium↗
Extracellular ATP triggers two functionally distinct calcium signalling pathways in PC12 cells.
We have investigated the effects of extracellular ATP on Ca2+ signalling, and its relationship to secretion in rat pheochromocytoma (PC12) cells. In single cells, extracellular ATP evoked two very distinct subcellular distributions of intracellular calcium concentration ([Ca2+]i), only one of which could be mimicked by the pyrimidine nucleotide UTP, suggesting the involvement of more than one cell surface receptor in mediating the ATP-induced responses. ATP and UTP were equipotent in activating a receptor leading to inositol phosphate production and the mobilisation of intracellular Ca2+. In some cells (19%) this rise in [Ca2+]i initiated at a discrete site and then propagated across the cell in the form of a Ca2+ wave. In addition to mobilising intracellular Ca2+ through a 'nucleotide' receptor sensitive to ATP and UTP, the results indicate that ATP also activates divalent cation entry through an independent receptor-operated channel. Firstly, ATP-induced entry of Ca2+ or Mn2+ was independent of Ca2+ mobilisation, as prior treatment of cell populations with UTP abolished the ATP-evoked release of intracellular Ca2+ stores, but left the Ca(2+)- and Mn(2+)-entry components uneffected. Secondly, although UTP and ATP were equally effective in generating inositol phosphates, only ATP stimulated divalent cation entry, indicating that ATP-activated influx was independent of phosphoinositide turnover. Thirdly, single cell experiments revealed a subpopulation of cells that responded to ATP with divalent cation entry without mobilising Ca2+ from intracellular stores. Lastly, the dihydropyridine antagonist, nifedipine, reduced the ATP-induced rise in [Ca2+]i by only 24%, suggesting that Ca2+ entry was largely independent of L-type voltage-operated Ca2+ channels. The Ca2+ signals could also be distinguished at a functional level. Activation of ATP-induced divalent cation influx was absolutely required to evoke transmitter release, because ATP triggered secretion of [3H]dopamine only in the presence of external Ca2+, and UTP was unable to promote secretion, irrespective of the extracellular [Ca2+]. The results suggest that the same extracellular stimulus can deliver different Ca2+ signals into the same cell by activating different Ca2+ signalling pathways, and that these Ca2+ signals can be functionally distinct.
Adenosine Triphosphate↗
A spermatid factor inhibits cAMP and calcium signaling in Sertoli but not Leydig cells.
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Animals↗
Calcium signalling in muscle. An international symposium held at Muskelzentrum, Universität Ulm, 5-7 October 1995.
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Animals↗
Calcium signaling in neurosecretory terminals and pituicytes.
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Animals↗
Source of nuclear calcium signals.
Transient increases of Ca2+ concentration in the nucleus regulate gene expression and other nuclear processes. We investigated whether nuclear Ca2+ signals could be regulated independently of the cytoplasm or were controlled by cytoplasmic Ca2+ signals. A fluorescent Ca2+ indicator that is targeted to the nucleus was synthesized by coupling a nuclear localization peptide to Calcium Green dextran, a 70-kDa Ca2+ indicator. Stimulation of rat basophilic leukemia cells by antigen or by photolytic uncaging of inositol 1,4,5-trisphosphate induced transient increases in nuclear and cytosolic Ca2+ concentrations. Elevations in the nuclear Ca2+ concentration followed those in the nearby perinuclear cytosol within 200 ms. Heparin-dextran, an inhibitor of the inositol 1,4,5-trisphosphate receptor that is excluded from the nucleus, was synthesized to specifically block the release of Ca2+ from cytosolic stores. Addition of this inhibitor suppressed Ca2+ transients in the nucleus and the cytosol. We conclude that the Ca2+ level in the nucleus is not independently controlled. Rather, nuclear Ca2+ increases follow cytosolic Ca2+ increases with a short delay most likely due to Ca2+ diffusion from the cytosol through the nuclear pores.
Amino Acid Sequence↗
Acetylcholine-induced calcium signaling associated with muscarinic receptor activation in cultured myenteric neurons.
BACKGROUND: Within the enteric nervous system, acetylcholine (ACh) is an important neurotransmitter. Experimental evidence has suggested that in myenteric neurons, calcium plays a key role in the coupling of cholinergic receptors to secretory responses. STUDY DESIGN: We investigated the effects of ACh on intracellular calcium concentration ([Ca2+]i) in individual myenteric neurons using fura-2 microspectrofluorometry. RESULTS: Resting [Ca2+]i in myenteric neurons was 62.5 +/- 3 nM. Acetylcholine produced dose-dependent increases in [Ca2+]i in myenteric neurons. As the concentration of ACh was increased from 0.1 to 100 microM, both the peak [Ca2+]i response as well as the percentage of responding neurons progressively increased, with a maximal effect at 100 microM (347 +/- 31 nM, 95 percent of neurons). The effect of ACh was not sensitive to pertussis toxin (100 ng/mL). Calcium ion (Ca2+) responses to ACh were abolished by removal of extracellular Ca2+ as well as exposure to nifedipine (10 microM). Characterization of the specific muscarinic subtype(s) involved in ACh-mediated Ca2+ transients was performed using the specific antagonists pirenzepine (M1), gallamine (M2), and 4-DAMP (M3). Pirenzepine (1 microM) blocked increases in [Ca2+]i induced by ACh; gallamine (1 microM) and 4-DAMP (1 microM) had no significant effect. Intracellular Ca2+ responses to ACh were not affected by incubation with the phorbol ester tetradecanoylphorbol-13-acetate (1 microM). CONCLUSIONS: These findings suggest that ACh induces increases in [Ca2+]i in myenteric neurons by promoting influx of extracellular Ca2+ through L-type voltage-dependent Ca2+ channels by activation of the M1 muscarinic receptor subtype. The Ca2+ response does not appear to involve a pertussis toxin-sensitive G protein.
Acetylcholine↗
Mutant analysis approaches to understanding calcium signal transduction through calmodulin and calmodulin regulated enzymes.
An example set of site-specific mutagenesis studies of calmodulin has been discussed in terms of strategy and how the results can provide insight into the functioning of calmodulin. A set of common examples for the study of calcium binding and enzyme activation were discussed. Essentially, site-specific mutagenesis in these initial studies is a perturbation approach. From these perturbation studies, structural features can be correlated in future studies with function and mechanisms of action proposed. More importantly, the approach allows efficient testing of proposed mechanisms and further probing of the molecular aspects of the signal transduction pathways. Clearly, the key functional feature that must be addressed in future studies is how the calcium binding steps in the mechanism are coupled to the enzyme activation step, which is the final step of the calmodulin-enzyme binding mechanism.
Amino Acid Sequence↗
Calcium signaling in endothelin- and platelet-derived growth factor-stimulated chondrocytes.
In bovine articular chondrocytes, endothelin (ET) and platelet-derived growth factor (PDGF) receptors mediate agonist-induced increases in inositol phosphate hydrolysis, cytoplasmic calcium concentration ([Ca2+]i), and mitogenesis. In most cells, ET stimulated nonoscillatory [Ca2+]i elevations with dose-dependent increases in both spike and plateau amplitudes. However, about 15% of cells showed oscillatory Ca2+ responses with a constant frequency and variable shape and duration of spiking. ET-1 and ET-2 were more potent than ET-3 in stimulating [Ca2+]i responses in inhibiting the specific binding of 125I-ET-1 and 125I-ET-3 and in promoting internalization of the receptor-ligand complex, consistent with actions through endothelin ETA receptors. Similar nonoscillatory and oscillatory patterns of Ca2+ responses were observed in PDGF-stimulated cells. In cells showing nonoscillatory Ca2+ responses to ET-1, subsequent stimulation with PDGF was frequently followed by the development of an oscillatory Ca2+ response. Nonoscillatory responses to both agonists were only slightly reduced in Ca(2+)-deficient medium, but the oscillatory responses were critically dependent on Ca2+ entry. Ca2+ spiking was not altered in the presence of the voltage-sensitive Ca2+ channel blocker, nifedipine; also, depolarization of chondrocytes by high K+ did not induce [Ca2+]i responses, confirming that voltage-sensitive calcium channels are not expressed in these cells. At high agonist concentrations, ET- but not PDGF-stimulated cells underwent rapid desensitization. Activation of ETA and PDGF receptors was associated with differential stimulation of thymidine incorporation; ET-1 induced a low-amplitude bell-shaped dose-response curve; PDGF induced a sustained sigmoidal and dose-dependent rise. These data indicate that two distinct types of Ca(2+)-mobilizing receptors initiate similar patterns of [Ca2+]i responses but have different capacities to maintain and reinitiate the Ca2+ signaling, as well as to promote mitogenesis.
Aging↗
Radiation-induced progressive decrease in fluid secretion in rat submandibular glands is related to decreased acinar volume and not impaired calcium signaling.
The mechanism(s) of radiation-induced salivary gland dysfunction is poorly understood. In the present study, we have assessed the secretory function (muscarinic agonist-stimulated saliva flow, intracellular calcium mobilization, Na+/K+/2Cl- cotransport activity) in rat submandibular glands 12 months postirradiation (single dose, 10 Gy). The morphological status of glands from control and irradiated rats was also determined. Pilocarpine-stimulated salivary flow was decreased by 67% at 12 months (but not at 3 months) after irradiation. This was associated with a 47% decrease in the wet weight of the irradiated glands. Histological and morphometric analysis demonstrated that acinar cells were smaller and occupied relatively less volume and convoluted granular tubules were smaller but occupied the same relative volume, while intercalated and striated ducts maintained their size but occupied a greater relative volume in submandibular glands from irradiated compared to control animals. In addition, no inflammation or fibrosis was observed in the irradiated tissues. Carbachol- or thapsigargin-stimulated mobilization of Ca2+ was similar in dispersed submandibular gland cells from control and irradiated animals. Further, [Ca2+]i imaging of individual ducts and acini from control and irradiated groups showed, for the first time, that mobilization of Ca2+ in either cell type was not altered by the radiation treatment. The carbachol-stimulated, bumetanide-sensitive component of the Na+/K+/ 2Cl- cotransport activity was also similar in submandibular gland cells from control and irradiated animals. These data demonstrate that a single dose of gamma radiation induces a progressive loss of submandibular gland tissue and function. This loss of salivary flow is not due to chronic inflammation or fibrosis of the gland or an alteration in the neurotransmitter signaling mechanism in the acinar or ductal cells. The radiation-induced decrease in fluid secretion appears to be related to a change in either the water-handling capacity of the acini or the number of acinar cells in the gland.
Animals↗
Lysosome-sarcoplasmic reticulum junctions. A trigger zone for calcium signaling by nicotinic acid adenine dinucleotide phosphate and endothelin-1.
Previous studies on pulmonary arterial smooth muscle cells have shown that nicotinic acid adenine dinucleotide phosphate (NAADP) evokes highly localized intracellular Ca(2+) signals by mobilizing thapsigargin-insensitive stores. Such localized Ca(2+) signals may initiate global Ca(2+) waves and contraction of the myocytes through the recruitment of ryanodine receptors on the sarcoplasmic reticulum via Ca(2+)-induced Ca(2+) release. Here we show that NAADP evokes localized Ca(2+) signals by mobilizing a bafilomycin A1-sensitive, lysosome-related Ca(2+) store. These lysosomal stores facilitate this process by co-localizing with a portion of the sarcoplasmic reticulum expressing ryanodine receptors to comprise a highly specialized trigger zone for NAADP-dependent Ca(2+) signaling by the vasoconstrictor hormone, endothelin-1. These findings further advance our understanding of how the spatial organization of discrete, organellar Ca(2+) stores may underpin the generation of differential Ca(2+) signaling patterns by different Ca(2+)-mobilizing messengers.
Animals↗
The Croonian lecture, 1988. Inositol lipids and calcium signalling.
The response of cells to many external stimuli requires a decoding process at the membrane to transduce information into intracellular messengers. A major decoding mechanism employed by a variety of hormones, neurotransmitters and growth factors depends on the hydrolysis of a unique inositol lipid to generate two key second messengers, diacylglycerol and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). Here I examine the second messenger function of Ins(1,4,5)P3 in controlling the mobilization of calcium. We know most about how this messenger releases calcium from internal reservoirs but less is known concerning the entry of external calcium. One interesting possibility is that Ins(1,4,5)P3 might function in conjunction with its metabolic product Ins(1,3,4,5)P4 to control calcium entry through a mechanism employing a region of the endoplasmic reticulum as a halfway house during the transfer of calcium from outside the cell into the cytoplasm. The endoplasmic reticulum interposed between the plasma membrane and the cytosol may function as a capacitor to insure against the cell being flooded with external calcium. When stimulated, cells often display remarkably uniform oscillations in intracellular calcium. At least two oscillatory patterns have been recognized suggesting the existence of separate mechanisms both of which may depend upon Ins(1,4,5)P3. In one mechanism, oscillations may be driven by periodic pulses of Ins(1,4,5)P3 produced by receptors under negative feedback control of protein kinase C. The other oscillatory mechanism may depend upon Ins(1,4,5)P3 unmasking a process of calcium-induced calcium release from the endoplasmic reticulum. The function of these calcium oscillations is still unknown. This Ins(1,4,5)P3/calcium signalling system is put to many uses during the life history of a cell.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗