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Recent work, stemming from genetic studies of phototransduction in Drosophila, has identified a set of homologous channel proteins that appear to mediate capacitative Ca2+ entry in species from worms and files to humans.
Synthetic calcium buffers, including fluorescent calcium indicators, were microinjected into squid 'giant' presynaptic nerve terminals to investigate the calcium signal that triggers neurotransmitter secretion. Digital imaging methods, applied in conjunction with the fluorescent calcium indicator dye fura-2, reveal that transient rises in presynaptic calcium concentration are associated with action potentials. Transmitter release terminates within 1-2 ms after a train of action potentials, even though presynaptic calcium concentration remains at micromolar levels for many seconds longer. Microinjection of the calcium buffer, EGTA, into the presynaptic terminal has no effect on transmitter release evoked by single presynaptic action potentials. EGTA injection does, however, block the change in calcium concentration measured by fura-2. Therefore, the calcium signal measured by fura-2 is not responsible for triggering release. These results suggest that the rise in presynaptic calcium concentration that triggers release must be highly localized to escape detection with fura-2 imaging. Unlike EGTA, microinjection of BAPTA--a calcium buffer with an equilibrium affinity for calcium similar to that of EGTA--produces a potent, dose-dependent, and reversible block of action-potential evoked transmitter release. The superior ability of BAPTA to block transmitter release apparently is due to the more rapid calcium-binding kinetics of BAPTA compared to EGTA. Because EGTA should bind calcium within a few tens of microseconds under the conditions of our experiments, the inability of EGTA to block release indicates that transmitter release is triggered within a few tens of microseconds after the entry of calcium into the presynaptic terminal.(ABSTRACT TRUNCATED AT 250 WORDS)
This study is to examine whether endothelial calcium signaling is different between atherosclerosis-prone thoracic aortas (TA) and atherosclerosis-resistant carotid arteries (CA) in normal rabbits and how it changes in early-stage atherosclerosis. Local endothelial calcium signaling was examined in arterial segments obtained from rabbits fed with normal or high-cholesterol diet for 1-4 weeks. Contrasting to normal CA, normal TA showed lower endothelial calcium signaling with more concentrated NF-kappaB in the endothelial nuclei. In the same hypercholesterolemic animal, fatty streak formation was much more prominent in TA than in CA. TA endothelial calcium signaling became augmented in the second week of hypercholesterolemia, being most pronounced in smooth regions adjacent to miniature fatty streaks. It was sporadically elevated even in regions away from any detectable TA fatty streak. When the entire TA was covered with fatty streaks in the fourth week of hypercholesteremia, endothelial calcium signaling returned to the original level. In comparison, CA endothelial calcium signaling was reduced around scattered fatty streaks. Reduced calcium signaling happened where CA fatty streaks were 150 microm long (covering 15-30 cells); and it extended to areas adjacent to larger fatty streaks. Moreover, NF-kappaB remained in the cytosol of endothelial cells covering CA fatty streaks. Our results indicate that inter-vascular differences in endothelial calcium signaling may provide partial explanation in their differential susceptibility in atherosclerosis.
An increase in cytoplasmic free calcium ion concentration is thought to play a critical role in the entry of resting T lymphocytes into the mitotic cycle. Not all murine T cells, however, generate such a calcium signal when exposed to mitogenic doses of concanavalin A (Con A), and the proportion of nonresponsive cells increases with age in adult mice. Since the frequency of T cells able to generate cytotoxic or lymphokine-secreting cells in culture also declines in old age, we have speculated that the defect in changes in Ca2+ concentration might underlie this functional deficit. To examine the relationship between functional competence and the calcium signaling pathway, we performed limiting dilution analyses of T cells enriched (by a fluorescence-based cell-sorting method) for high or low calcium signal generation. We found that rapid Ca2+ signal generation after Con A exposure did indeed correlate well with functional competence in Con A-stimulated limiting dilution cultures. Furthermore, selection for Ca2+ signals induced by anti-CD3 antibody, or even by ionomycin, similarly enriched for the functionally competent set of T cells. The ionomycin result, in particular, suggests that the difference between the reactive and nonreactive sets of T cells is unlikely to result simply from alterations in receptor-linked signal transduction pathways. We discuss a model in which immune senescence involves an age-dependent increase in a subset of T cells in which changes in cytoplasmic Ca2+ concentration are relatively difficult to induce by either receptor-dependent mitogens or ionophores.
The mechanisms of glutamate-induced changes in intracellular free calcium concentration in Bergmann glial cells in mouse cerebellar slices were investigated by Fura-2-based microfluorimetry. Extracellular applications of glutamate, quisqualate and kainate triggered an increase in cytoplasmic calcium concentration, whereas N-methyl-D-aspartate and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate were ineffective. The calcium elevation triggered by kainate was completely blocked by removal of calcium ions from the external solutions or by slice incubation with 6-cyano-7-nitroquinoxaline-2,3-dione. Conversely, both glutamate- and quisqualate-induced intracellular calcium transients were only slightly attenuated by slice incubation with either 6-cyano-7-nitroquinoxaline-2,3-dione or calcium-free solution, suggesting the intracellular origin for calcium ions. The glutamate-triggered cytosolic calcium increases were inhibited by slice incubation with thapsigargin, the inhibitor of intracellular calcium pumps, or by intracellular perfusion of Bergmann glial cells with heparin, the antagonist of inositol-1,4,5-trisphosphate-gated calcium release channels. Therefore the calcium release from inositol-1,4,5-trisphosphate-sensitive intracellular stores plays the major role in glutamate-induced calcium signalling. We concluded that Bergmann glial cells express calcium permeable ionotropic glutamate receptors, which might be important for generation of fast calcium signals. However, slow glutamate-evoked calcium signals are mostly determined by inositol-1,4,5-trisphosphate-dependent intracellular signalling chain.
BACKGROUND & AIMS: Acetylcholine-induced calcium signaling dynamics have been described in cell monolayers derived from colonic mucosa, but not in intact colonic crypts. The aim of this study was to characterize the spatiotemporal characteristics of calcium signaling induced by acetylcholine in isolated intact rat colonic crypts and to identify the muscarinic receptor subtype coupled to this signaling pathway. METHODS: Isolated crypts from the distal colon of male Wistar rats were loaded with the calcium-sensitive dye Fura-2 and imaged with a charge-coupled device video camera. RESULTS: Acetylcholine mobilized intracellular calcium with an EC50 of 3.9 micromol/L. The response was initiated at the base of the crypt and progressed toward the surface. The velocity of propagation was dose dependent. Addition of muscarinic antagonists inhibited the response (pKb values calculated for pirenzepine and 4-DAMP, 6.08 and 8.65, respectively). Microperfusion of acetylcholine initiated a calcium signal throughout the lower half of the crypt. Microinjection of inositol 1,4,5-triphosphate induced a propagation of a calcium signal along the crypt axis. Heptanol inhibited the velocity of acetylcholine-induced wave propagation by 33%. CONCLUSIONS: M3 muscarinic receptors are coupled to the mobilization of calcium from intracellular stores of intact, isolated rat colonic crypts. Intercellular communication potentiates the propagation of the acetylcholine-induced calcium signal along the crypt axis.
Calcium signaling, one of the most widespread signaling mechanisms in cells, is generally carried out by EF-hand proteins, characterized by a helix-loop-helix motif paired in functional domains. EF-hand proteins may be viewed as molecular switches activated by calcium concentration transients. The EF-hand structural database has grown to a point where meaningful inferences on the functional conformational rearrangements upon calcium binding can be made by comparing a fair number of pairs of end points, i.e., the structures of the apo and calcium-bound forms. More compact descriptors of the movement associated with calcium binding, in terms of principal component analysis of the six interhelical angles, have also become available. Dynamic information obtained by NMR, also with the aid of calcium substitution with paramagnetic lanthanides, is shedding light on the intrinsic amplitude of the conformational degrees of freedom sampled by the various members of the EF-hand superfamily, as well as on the time scales of the motions. Particularly, NMR of lanthanide derivatives helps in capturing long time scale motions. Both static and dynamic pictures reveal a large variety of behaviors. It is increasingly recognized that the EF-hand machinery has differentiated its behavior during evolution in several ways, e.g., by modifying one of the loops, by undergoing a further duplication after the initial motif duplication that originated the functional domain, or by acquiring the ability to dimerize.
The synthesis and biological evaluation of ethyl 5-amino-4-(3-pyridyl)-2-methyl-6,7,8,9-tetrahydro-4H-pyrano[2,3-b]quinoline-3-carboxylates (9-11) is described. We have found that these compounds inhibit AChE with a mild potency, mitigates the [Ca(2+)](c) triggered by high K(+), and cause neuroprotection against Ca(2+) overloading and free radical-induced neuronal death.
The antiestrogen tamoxifen is commonly used to treat breast cancer, but it also has therapeutic activity in several other types of cancer. Many of these tumors, including malignant gliomas, are estrogen receptor negative. Nonetheless, high concentrations of tamoxifen can directly reduce cell proliferation in some of these tumors and induce apoptosis. In this study, the role of tamoxifen in calcium signaling and calcium-induced cell death was studied in both malignant glioma cell lines and MCF-7 breast cancer cells. Tamoxifen potently increased the spatial expansion of calcium waves by 30-150% while significantly enhancing and prolonging agonist-induced calcium elevations. Furthermore, tamoxifen pretreatment accelerated calcium ionophore-induced death by more than 20 min, suggesting that tamoxifen lowered cellular resistance to calcium loads. In contrast to its potentiating of calcium signaling in tumors, tamoxifen had no significant effect on calcium signaling in cultures of primary astrocytes from either human or rat brain. This study demonstrates the existence of calcium signaling in breast cancer and glioma cells and identifies tamoxifen as a potential modulator of tumor-associated calcium signaling.
Calcium influx into lymphocytes is essential for activation, differentiation, and effector functions. While several channel- and receptor-types contribute to calcium influx, voltage-gated calcium channels (VGCC) mediate a well-characterized calcium influx pathway that is most exclusively identified in excitable cells. The role of L-type VGCCs, which belong to high-voltage activated calcium channels and are defined as dihydropyridine (DHP) receptors in excitable cells, is well documented. Interestingly, while lymphocytes do not range in the excitable cell category, the modulatory role of DHP agonists and antagonists and the identification of L-type VGCC-related molecules in B and T lymphocytes, mainly in Th2 cells, suggest these proteins are involved in the calcium response of these cells. Because the identity and the regulation of DHP receptors/channels in lymphocytes is far from being solved, we will discuss the challenging issues of demonstrating a role of L-type VGCCs in nonexcitable cells and the arguments supporting their role in lymphocytes. We will comment on the limitation of the use of DHP agonists and antagonists to ascertain a specific involvement of L-type VGCCs in lymphocyte calcium signaling. Finally, we will provide new clues on the interest of a potential use of DHP antagonists in Th2-cell-mediated pathology.
Mitogen-induced increases in free calcium ion concentration ([Ca]i) are a key element of the process by which T lymphocytes are induced to proliferate and differentiate into effector cells. T cells from old mice exhibit lower average rises in calcium concentration than T cells from young donors when stimulated with either mitogenic lectins or antibodies to the CD3 chains of the antigen receptor. The decline with age in calcium signal generation is largely attributable to a shift from naïve to memory T cells, in that memory T cells, from mice of any age, are more resistant to mitogen-induced changes in [Ca]i. The decline in calcium signal generation is likely to be functionally significant, since T cells isolated on the basis of poor calcium signals show diminished ability to produce and to respond to the growth factor IL-2. Con A induces a transient increase in uptake of radiolabeled calcium from extracellular sources, and the extent of this increase declines with age. Alterations in production of inositol tris-phosphate (IP3) seem not to contribute to age-related changes in calcium signal generation. T cells from old mice, and memory T cells from mice of any age, are relatively resistant to increases in [Ca]i even when these are induced by receptor-independent stimuli such as the calcium ionophore ionomycin. The ionomycin-resistance of memory T cells suggests that these cells may have an augmented ability to buffer changes in [Ca]i, perhaps by increased activity of the ATP-dependent plasma membrane calcium pump. It seems likely that age-related declines in calcium signal generation contribute to the functional immunodeficiency of old age.
Studies have been performed to understand the interactions and the role which intracellular calcium and intracellular pH have in mediating mitogen-stimulated cellular proliferation. Stimulation of Syrian hamster embryo (SHE) cells with the mitogen platelet-derived growth factor A/B (PDGF) results in intracellular acidification and capacitative calcium entry involving the intracellular release of calcium via the inositol trisphosphate gamma receptor calcium channel, followed by an extracellular influx of calcium through a dihydropyridine-sensitive plasma membrane calcium channel. Chronic extracellular/intracellular acidification results in the inactivation of both these calcium channels due to slowly reversible protein alterations. Paradoxically, transient intracellular acidification, like that following PDGF stimulation, could not stimulate the activation of either calcium channel. In addition, even though intracellular calcium fluxes by themselves could intiate intracellular acidification, loss of the PDGF-induced calcium signal did not result in the loss of the PDGF-induced transient intracellular acidification. Importantly with regard to the role intracellular calcium and pH have in mediating the mitogenic signal leading to cellular proliferation, chronic extracellular/intracellular acidification, which leads to a complete loss of the PDGF-induced calcium signal, did not result in the loss of PDGF-induced mitogenesis. These results indicate that the PDGF-induced calcium signal is not essential for PDGF-stimulated mitogenesis in Syrian hamster embryo cells. In contrast, blocking the PDGF-induced transient intracellular acidification completely blocks PDGF-induced mitogenesis, indicating that the mitogen-induced transient intracellular acidification, unlike the intracellular calcium ion signal, is indispensible for cellular proliferation in Syrian hamster embryo cells.
Signaling between osteoblasts and osteoclasts is important in bone homeostasis. We previously showed that human osteoblasts propagate intercellular calcium signals via two mechanisms: autocrine activation of P2Y receptors, and gap junctional communication. In the current work we identified mechanically induced intercellular calcium signaling between osteoblasts and osteoclasts and among osteoclasts. Intercellular calcium responses in osteoclasts required P2 receptor activation but not gap junctional communication. Pharmacological studies and reverse transcriptase-PCR amplification demonstrated that human osteoclasts expressed functional P2Y1 receptors, but, unexpectedly, desensitization of P2Y1 did not block calcium signaling to osteoclasts. We also found that osteoclasts expressed functional P2X7 receptors and showed that pharmacological inhibition of these receptors blocked calcium signaling to osteoclasts. Thus these studies show that calcium signaling between osteoblasts and osteoclasts occurs via activation of P2 receptors, but that different families of P2 receptors are required for calcium signaling in these two cell types. Intercellular calcium signaling among bone cells is therefore amenable to pharmacological manipulation that will specifically affect only bone-forming or bone-resorbing cells. P2 receptors may be important drug targets for the modulation of bone turnover.
Calcium is central in the regulation of cardiac contractility, growth and gene expression. Variations in the amplitude, frequency and compartmentalization of calcium signals are decoded by calcium/calmodulin-dependent enzymes, ion channels and transcription factors. Understanding the circuitry for calcium signaling creates opportunities for pharmacological modification of cardiac function.
Glial calcium signals play important roles during CNS development. Calcium transients induced by ATP, acting on purinergic receptors, stimulate DNA synthesis, increase astrocytic and neural stem cell proliferation, and are prominent during the differentiation of radial glia. We have shown previously that expression of P2Y receptors in astrocytes is altered when connexin43 (Cx43) is downregulated. To evaluate the consequences of Cx43 deletion on calcium signaling during neural progenitor development, studies were performed on neurospheres derived from embryonic striatum. After adhesion, cells migrating from wild-type (WT) and Cx43-null neurospheres displayed spontaneous calcium oscillations. Such activity was blunted by apyrase, 2'-deoxy-N6-methyladenosine 3',5'-bisphosphate (MRS-2179), and suramin, suggesting that ATP released by neural cells acts on purinergic receptors to induce calcium oscillations. The amplitudes of Ca2+ transients induced by P2Y but not P2X receptor agonists were larger in WT than in Cx43-null progenitors, suggesting that these two cell populations express different P2 receptors. Suramin, a nonselective P2 receptor antagonist, and MRS-2179, a P2Y1 receptor-selective antagonist, reduced the proliferation rate and the migration of WT progenitor cells to levels similar to those of Cx43-null cells. Conversely, exogenous expression of P2Y1 receptors in Cx43-null cells restored their migration pattern to levels seen in WT progenitors. However, treatment with P2 receptor antagonists did not alter the ratio of nestin to GFAP expression in WT neural progenitors. These data show that altered autocrine-paracrine communication attributable to reduced levels of P2Y1 receptors in neural progenitor cells lacking Cx43 affects proliferation and migration but not cell differentiation during early CNS development.
1. Otilonium bromide (OB) is a smooth muscle relaxant used in the treatment of irritable bowel syndrome. Otilonium bromide has been shown to interfere with the mobilization of calcium in intestinal smooth muscle, but the effects on other intestinal tissues have not been investigated. We identified the muscarinic receptor subtype coupled to calcium signals in colonic crypt derived from the human colonic epithelium and evaluated the inhibitory effects of OB. 2. Calcium signals were monitored by fluorescence imaging of isolated human colonic crypts and Chinese hamster ovary cells stably expressing the cloned human muscarinic M(3) receptor subtype (CHO-M(3)). Colonic crypt receptor expression was investigated by pharmacological and immunohistochemical techniques. 3. The secretagogue acetylcholine (ACh) stimulated calcium mobilization from intracellular calcium stores at the base of human colonic crypts with an EC(50) of 14 micro M. The muscarinic receptor antagonists 4-DAMP, AF-DX 384, pirenzepine and methroctamine inhibited the ACh-induced calcium signal with the following respective IC(50) (pK(b)) values: 0.78 nM (9.1), 69 nM (7.2), 128 nM (7.1), and 2510 nM (5.8). 4. Immunohistochemical analyses of muscarinic receptor expression demonstrated the presence of M(3) receptor subtype expression at the crypt-base. 5. Otilonium bromide inhibited the generation of ACh-induced calcium signals in a dose dependent manner (IC(50)=880 nM). 6. In CHO-M(3) cells, OB inhibited calcium signals induced by ACh, but not ATP. In addition, OB did not inhibit histamine-induced colonic crypt calcium signals. 7. The present studies have demonstrated that OB inhibited M(3) receptor-coupled calcium signals in human colonic crypts and CHO-M(3) cells, but not those induced by stimulation of other endogenous receptor types. We propose that the M(3) receptor-coupled calcium signalling pathway is directly targeted by OB at the level of the colonic epithelium, suggestive of an anti-secretory action in IBS patients suffering with diarrhoea.
Agonist-induced cytoplasmic calcium signals often have profound effects on the membrane potential during cellular activation. In the present study, we report that cytoplasmic calcium elevation can regulate the membrane potential by a novel mechanism. TRESK, a recently described member of the two-pore domain potassium (2PK(+)) channel family, was activated 5-15-fold after stimulation of various Ca(2+)-mobilizing receptors in Xenopus oocytes. Extracellular application of ionomycin, as well as the microinjection of inositol 1,4,5-trisphosphate or calcium, also evoked TRESK activation, whereas microinjection of EGTA or pretreatment of the oocytes with thapsigargin prevented the receptor-mediated effect. These data indicate that TRESK is activated by increased cytoplasmic calcium concentration. However, application of Ca(2+) to inside-out membrane patches failed to influence TRESK single channel activity, suggesting that cytoplasmic factors are also required for the regulation. Cyclosporin A and FK506, specific inhibitors of the calcium/calmodulin-dependent protein phosphatase (calcineurin), completely eliminated TRESK activation. Coexpression of a constitutively active form of calcineurin with TRESK increased the basal background K(+) current and attenuated the response of the channel to the calcium signal, indicating that TRESK was activated by the permanent calcineurin activity. Serine 276 was identified as the major functional target of calcineurin in TRESK by alanine-scanning mutagenesis. This is the first example of calcineurin being involved in the regulation of a two-pore domain K(+) channel, and thus, TRESK channels may regulate the excitability of neurons and other cell types in response to Ca(2+)-mobilizing hormones and neurotransmitters in a manner that is sensitive to immunosuppressive drugs.
All secretory anterior pituitary cells exhibit spontaneous and extracellular calcium-dependent electrical activity, but differ with respect to the patterns of firing and associated calcium signaling and hormone secretion. Thus, somatotrophs and lactotrophs fire plateau-bursting action potentials spontaneously and without coupling to calcium release from intracellular stores, which generate calcium signals of sufficient amplitude to keep steady hormone release. In these cells, both spontaneous electrical activity and basal hormone secretion can be further amplified by activation of Gq/11 and Gs-coupled receptors and inhibited by Gi/o/z-coupled receptors. In contrast, gonadotrophs fire single, high-amplitude spikes with limited ability to promote calcium influx and exocytosis, whereas activated Gq/11-coupled receptors in these cells transform single-action potential spiking into the plateau-bursting type of electrical activity and trigger periodic high-amplitude calcium signals and exocytosis of prestored secretory vesicles. Here, we review biochemical and biophysical aspects of spontaneous and receptor-controlled electrical activity, calcium signaling, and hormone secretion in pituitary cells.