L-histidine sensing by calcium sensing receptor inhibits voltage-dependent calcium channel activity and insulin secretion in β-cells

AimsOur goal was to test the hypothesis that the histidine-induced activation of calcium sensing receptor (CaR) can regulate calcium channel activity of L-type voltage dependent calcium channel (VDCC) due to increased spatial interaction between CaR and VDCC in β-cells and thus modulate glucose-induced insulin secretion.Main methodsRat insulinoma (RINr1046-38) insulin-producing β-cells were cultured in RPMI-1640 medium on 25 mm diameter glass coverslips in six-well culture plates in a 5% CO₂ incubator at 37 °C. The intracellular calcium concentration, [Ca²⁺]_i, was determined by ratio fluorescence microscopy using Fura-2AM. The spatial interactions between CaR and L-type VDCC in β-cells were measured by immunofluorescence confocal microscopy using a Nikon C1 laser scanning confocal microscope. The insulin release was determined by enzyme-linked immunosorbent assay (ELISA).Key findingsThe addition of increasing concentrations of L-histidine along with 10 mM glucose resulted in 57% decrease in [Ca²⁺]_i. The confocal fluorescence imaging data showed 5.59 to 8.62-fold increase in colocalization correlation coefficient between CaR and VDCC in β-cells exposed to L-histidine thereby indicating increased membrane delimited spatial interactions between these two membrane proteins. The insulin ELISA data showed 54% decrease in the 1st phase of glucose-induced insulin secretion in β-cells exposed to increasing concentrations of L-histidine.SignificanceL-histidine-induced increased spatial interaction of CaR with VDCC can inhibit calcium channel activity of VDCC and consequently regulate glucose-induced insulin secretion by β-cells. The L-type VDCC could therefore be a potential therapeutic target in diabetes.     

GTPγS-induced calcium transients and exocytosis in human neutrophils

Exocytosis and intracellular free calcium ([Ca²⁺]_in) were simultaneously recorded in single human neutrophils using patch-clamp capacitance measurements and the fura-2 fluorescence ratio method. Intracellular application of guanosine-5-O(3-thiotriphosphate) (GTPS) stimulates both exocytosis and a calcium transient. The calcium transient starts to develop after a lag phase of 40s and normally appears to trigger the onset of exocytosis indicated by the beginning of the capacitance increase. After this delay [Ca²⁺]_in increases from 150 nM to 600 nM with a sigmoidal time course. The peak concentration is reached within 30 s but the main increase occurs during 3s. [Ca²⁺]_in subsequently decays within 1–2 min to a level which is close to the resting value. This calcium transient is due to calcium release from inositoltrisphosphate-sensitive intracellular stores. Exocytosis also occurs if the calcium transient is abolished by intracellular EGTA but the lag phase is markedly prolonged. The GTPS-induced calcium transient is very similar to that observed after stimulation with N-formyl-methionyl-leucyl-phenylalanine. The interplay between guanine nucleotides, [Ca²⁺]_in and exocytosis in neutrophils closely resembles previous results obtained in mast cells suggesting a similar regulation of exocytosis in both cell types.     

How do calcium channels transport calcium ions?

Calcium channel activity is crucial for many fundamental physiological processes ranging from the heart beat to synaptic transmission. The channel-forming protein, of about 2000 amino acids, comprises four domains internally homologous to each other. Voltage-dependent Ca2+ channels are the most selective ion channels known. Under physiological conditions, they prefer Ca2+ over Na+ by a ratio of about 1000:1. To explain at the same time the exquisite ion selectivity and the large Ca2+ ion turnover rate of Ca2+ channels (approximately 3 x 10(6) ions/s), two kind models have been proposed. In one, the conduction pathway possesses two high-affinity binding sites. When two Ca2+ ions are bound to each site, the mutual repulsion between them speeds the exit rate for the ions, causing greater ion permeation through the pore. The second model hypothesizes the existence of a single site having a charged structure able to attract multiple, interacting ions, simultaneously. Recent studies that combine mutagenesis and electrophysiology show that the high-affinity binding site is formed by a ring of glutamate residues located in the pore forming region of the Ca2+ channel. As proposed in the second class of models, the results suggest that four glutamate residues, one glutamate donated by each repeat, combine to form a single high-affinity site. In this review the different conduction models for Ca2+ channels are discussed and confronted with structural data.     

Do calcium and calmodulin trigger maturation in amphibian oocytes?

In contrast to earlier reports (J. L. Maller and E. G. Krebs, 1980, Curr. Top. Cell. Regul. 16, 271-311; M. Moreau, J. P. Vilian, and P. Guerrier, 1980, Dev. Biol. 78, 201-214; W. J. Wasserman and L. D. Smith, 1981, J. Cell Biol. 89, 389-394; D. Huchon, R. Ozon, E. H. Fischer, and J. G. Demaille, 1981, Mol. Cell. Endocrinol. 22, 211-222) calmodulin preparations isolated from Xenopus laevis ovaries or obtained commercially rarely induced maturation upon microinjection into individual oocytes. Calmodulin injections did result in significant cases of maturation when oocytes were first pretreated (primed) with calcium-free (EGTA) OR-2 and then injected in regular OR-2 medium. However, under these conditions the injected buffer solution alone was sometimes found to induce maturation. Under more optimal priming conditions, cases were found where as high as 100% of the oocytes matured simply by returning them to regular OR-2 medium. To determine which divalent cations could be involved in the priming effects of EGTA pretreatment we repeated the earlier ionophore work of W. J. Wasserman and Y. Masui (1975, J. Exp. Zool. 193, 369-375), looking not just at calcium and magnesium but other divalent cations as well. Several divalent cations (10 mM) were found to induce germinal vesicle breakdown with the following tentative order of efficacy, Co2+ greater than or equal to Zn2+ greater than or equal to Mn2+ greater than Ca2+ greater than Mg2+ greater than Ba2+, regardless of whether or not ionophore A23187 was present. These results, along with other reports in the literature, are discussed with respect to the theory that a rise in free calcium and calmodulin is involved in triggering oocyte maturation; we conclude that neither is involved.     

Is calpain activity regulated by membranes and autolysis or by calcium and calpastatin?

Although the Ca(2+)-dependent proteinase (calpain) system has been found in every vertebrate cell that has been examined for its presence and has been detected in Drosophila and parasites, the physiological function(s) of this system remains unclear. Calpain activity has been associated with cleavages that alter regulation of various enzyme activities, with remodeling or disassembly of the cell cytoskeleton, and with cleavages of hormone receptors. The mechanism regulating activity of the calpain system in vivo also is unknown. It has been proposed that binding of the calpains to phospholipid in a cell membrane lowers the Ca2+ concentration, [Ca2+], required for the calpains to autolyze, and that autolysis converts an inactive proenzyme into an active protease. Recent studies, however, show that the calpains bind to specific proteins and not to phospholipids, and that binding to cell membranes does not affect the [Ca2+] required for autolysis. It seems likely that calpain activity is regulated by binding of Ca2+ to specific sites on the calpain molecule, with binding to each site eliciting a response (proteolytic activity, calpastatin binding, etc.) specific for that site. Regulation must also involve an, as yet, undiscovered mechanism that increases the affinity of the Ca(2+)-binding sites for Ca2+.     

Regulation of calcium in pancreatic α- and β-cells in health and disease

The glucoregulatory hormones insulin and glucagon are released from the β- and α-cells of the pancreatic islets. In both cell types, secretion is secondary to firing of action potentials, Ca(2+)-influx via voltage-gated Ca(2+)-channels, elevation of [Ca(2+)](i) and initiation of Ca(2+)-dependent exocytosis. Here we discuss the mechanisms that underlie the reciprocal regulation of insulin and glucagon secretion by changes in plasma glucose, the roles played by different types of voltage-gated Ca(2+)-channel present in α- and β-cells and the modulation of hormone secretion by Ca(2+)-dependent and -independent processes. We also consider how subtle changes in Ca(2+)-signalling may have profound impact on β-cell performance and increase risk of developing type-2 diabetes.     

Amyloid β-peptide directly induces spontaneous calcium transients,delayed intercellular calcium waves and gliosis in rat cortical astrocytes

The contribution of astrocytes to the pathophysiology of AD (Alzheimer''s disease) and the molecular and signalling mechanisms that potentially underlie them are still very poorly understood. However, there is mounting evidence that calcium dysregulation in astrocytes may be playing a key role. Intercellular calcium waves in astrocyte networks in vitro can be mechanically induced after Aβ (amyloid β-peptide) treatment, and spontaneously forming intercellular calcium waves have recently been shown in vivo in an APP (amyloid precursor protein)/PS1 (presenilin 1) Alzheimer''s transgenic mouse model. However, spontaneous intercellular calcium transients and waves have not been observed in vitro in isolated astrocyte cultures in response to direct Aβ stimulation in the absence of potentially confounding signalling from other cell types. Here, we show that Aβ alone at relatively low concentrations is directly able to induce intracellular calcium transients and spontaneous intercellular calcium waves in isolated astrocytes in purified cultures, raising the possibility of a potential direct effect of Aβ exposure on astrocytes in vivo in the Alzheimer''s brain. Waves did not occur immediately after Aβ treatment, but were delayed by many minutes before spontaneously forming, suggesting that intracellular signalling mechanisms required sufficient time to activate before intercellular effects at the network level become evident. Furthermore, the dynamics of intercellular calcium waves were heterogeneous, with distinct radial or longitudinal propagation orientations. Lastly, we also show that changes in the expression levels of the intermediate filament proteins GFAP (glial fibrillary acidic protein) and S100B are affected by Aβ-induced calcium changes differently, with GFAP being more dependent on calcium levels than S100B.     

Correlation between inhibition of calcium—dependent apoptosis by cyclosporin A and calcium transportation in HL—60 cells

Both calcium ionophore A23187 and endoplasmic reticulum Ca^2 -ATPase inhibitor thapsigargin (Tg) could increse intracellular free calcium concentration and induce apoptosis in some cell lines.In the present study,we found that HL-60 cells treated with A23187 (1μg/ml) for 4h or with Tg(0.5μg/ml) for 2h showed typical characteristics of apoptosis.Pretreatment with nontoxic concentration of cyclosporin A (CsA) (1μg/ml) could block these effects.Flow cytometric analysis of intracellular Ca^2 after staining with fluo-3 AM showed that CsA did not prevent the increase of intracellular calcium induced by A23187 or Tg,but it could maintain the high level of intracellular Ca^2 for a long time.These results suggest that CsA may prevent calcium-induced apoptosis by blocking the transportation of Ca^2 in HL-60 cells.     

Are auxin and calcium movements in corn coleoptiles linked processes?

In this paper we present evidence for a tight linkage of calcium and auxin fluxes in corn coleoptile tissue. Imposed lateral gradients of auxin concentration induce a movement of calcium ions to the side of lower auxin concentration in the tissue. Imposing a lateral gradient of calcium concentration in turn leads to a movement of auxin to the side of lower calcium concentration. Whether these fluxes are using symplastic or apoplastic pathways or are transcellular cannot be decided from the results.Countercurrent fluxes of calcium ions and auxin could be demonstrated in polar transport experiments. This might be a further indication of a complex carrier system in the plasma membrane extruding auxin out of the cell with a concomitant calcium influx from the cell wall space into the cytoplasm.     

T-type calcium channel blockers: spiro-piperidine azetidines and azetidinones—optimization,design and synthesis

A series of spiro-azetidines and azetidinones has been evaluated as novel blockers of the T-type calcium channel (Ca_V3.2) which is a new therapeutic target for the potential treatment of both inflammatory and neuropathic pain. Confirmation and optimization of the potency, selectivity and DMPK properties of leads will be described.     

The calcium sensing receptor: from calcium sensing to signaling

The Ca2+-sensing receptor(the Ca SR),a G-protein-coupled receptor,regulates Ca2+ homeostasis in the body by monitoring extracellular levels of Ca2+([Ca2+]o) and responding to a diverse array of stimuli.Mutations in the Ca2+-sensing receptor result in hypercalcemic or hypocalcemic disorders,such as familial hypocalciuric hypercalcemia,neonatal severe primary hyperparathyroidism,and autosomal dominant hypocalcemic hypercalciuria.Compelling evidence suggests that the Ca SR plays multiple roles extending well beyond not only regulating the level of extracellular Ca2+ in the human body,but also controlling a diverse range of biological processes.In this review,we focus on the structural biology of the Ca SR,the ligand interaction sites as well as their relevance to the disease associated mutations.This systematic summary will provide a comprehensive exploration of how the Ca SR integrates extracellular Ca2+ into intracellular Ca2+ signaling.     

Structural basis for calcium and phosphatidylserine regulation of phospholipase C δ1

Many membrane-associated enzymes, including those of the phospholipase C (PLC) superfamily, are regulated by specific interactions with lipids. Previously, we have shown that the C2 domain of PLC δ1 is required for phosphatidylserine (PS)-dependent enzyme activation and that activation requires the presence of Ca(2+). To identify the site of interaction and the role of Ca(2+) in the activation mechanism, we mutagenized three highly conserved Ca(2+) binding residues (Asp-653, Asp-706, and Asp-708) to Gly in the C2 domain of PLC δ1. The PS-dependent Ca(2+) binding affinities of the mutant enzymes D653G, D706G, and D708G were reduced by 1 order of magnitude, and the maximal level of Ca(2+) binding was reduced to half of that of the native enzyme. The level of Ca(2+)-dependent PS binding was also reduced in the mutant enzymes. Under basal conditions, the Ca(2+) dependence and the maximal level of hydrolysis of phosphatidylinositol 4,5-bisphosphate were not altered in the mutants. However, the Ca(2+)-dependent PS stimulation was severely defective. PS reduces the K(m) of the native enzyme almost 20-fold, but far less for the mutants. Replacing Asp-653, Asp-706, and Asp-708 simultaneously with glycine in the C2 domain of PLC δ1 leads to a complete and selective loss of the stimulation and binding by PS. These results show that D653, D706, and D708 are required for Ca(2+) binding in the C2 domain and demonstrate a mechanism by which C2 domains can mediate regulation of enzyme activity by specific lipid ligands.     

The role of calcium in chloroplasts—an intriguing and unresolved puzzle

More than 70?years of studies have indicated that chloroplasts contain a significant amount of calcium, are a potential storage compartment for this ion, and might themselves be prone to calcium regulation. Many of these studies have been performed on the photosynthetic light reaction as well as CO(2) fixation via the Calvin-Benson-Bassham cycle, and they showed that calcium is required in several steps of these processes. Further studies have indicated that calcium is involved in other chloroplast functions that are not directly related to photosynthesis and that there is a calcium-dependent regulation similar to cytoplasmic calcium signal transduction. Nevertheless, the precise role that calcium has as a functional and regulatory component of chloroplast processes remains enigmatic. Calcium concentrations in different chloroplast subcompartments have been measured, but the extent and direction of intra-plastidal calcium fluxes or calcium transport into and from the cytosol are not yet very well understood. In this review we want to give an overview over the current knowledge on the relationship between chloroplasts and calcium and discuss questions that need to be addressed in future research.     

High accumulations of calcium and phosphorus in women’s pubic symphysis

To elucidate compositional changes of the pubic symphysis (PS) by aging, elements of pubic symphyses (PSs) removed from 26 cadavers were determined by inductively coupled plasma atomic-emission spectrometry. It was found that the relative contents (RCs) of calcium and phosphorus in women’s PSs were about three-and five-fold amounts as compared with those in men’s PSs, respectively. In contrast, the RCs of sulfur, magnesium, sodium, and iron in women’s PSs were somewhat lower than those in men’s PSs. The accumulations of calcium (Ca) and phosphorus (P) in women’s PSs occurred mainly beyond the age of 70-yr-old, but did not occur in men’s PSs.     

Regulation and molecular mechanisms of calcium transport genes: do they play a role in calcium transport in the uterine endometrium?

Maintenance of calcium (Ca) balance in the uterus is critically important for many physiological functions, including smooth muscle contraction during embryo implantation. Ca transport genes, i.e., transient receptor potential cation channel subfamily V members 5/6 (TRPV5/6), calbindins, plasma membrane Ca(2+)-ATPase 1 (PMCA1), and NCX1/NCKX3, may play roles in the uterus for Ca transport and reproductive function. Although these Ca transport genes may have a role in Ca metabolism, their role(s) and molecular mechanisms require further elucidation. In this review, we highlight the expression and regulation of Ca transport genes in the uterus to clarify their potential role(s). Since Ca transport genes are abundantly expressed in reproductive tissues in a distinct manner, they may be involved in specific uterine functions including fetal implantation, Ca homeostasis, and endometrial cell production.     

Antiarrhythmic drug amiodarone displays antifungal activity,induces irregular calcium response and intracellular acidification of Aspergillus niger – Amiodarone targets calcium and pH homeostasis of A. niger

The rapidly developing resistance of fungi to antifungal drugs is a serious health problem. Today’s drugs mainly target cell membrane composition and synthesis. Moreover, some of them have serious side effects. New antifungal drugs targeting different molecular pathways are necessary. Amiodarone, an FDA approved antiarrhythmic drug displays antifungal activity. It targets calcium and pH homeostasis. In concentrations above 25 μM, it inhibits the growth of the filamentous fungi Aspergillus niger. It triggers a biphasic calcium response accompanied by a high [Ca²⁺]_c resting level and an intracellular acidification from 7.5 to 6.0, both of which are concentration dependent. Both extracellular calcium and calcium from intracellular organelles are sources of the transient second cytosolic calcium peak, whose amplitude is 0.12 μM for cells treated with 0.1 mM amiodarone. In P-type ATPase deficient A. niger strains pmrAΔ and pmcAΔ, the [Ca²⁺]_c resting level after amiodarone treatment is at least twice as high as that of the wild type, which correlates with fungal viability and hypersensitivity to amiodarone. A combination of amiodarone and amphotericin B is additive in terms of cell viability and cytosolic calcium influx. In contrast, a combination of azole drugs and amiodarone has a synergistic effect on the viability of fungi.     

Is the osteoclast calcium "receptor" a receptor-operated calcium channel?

Elevated extracellular calcium levels ([Ca2+]e) inhibit osteoclast function by elevating cytosolic free calcium levels ([Ca2+]i), presumably via the activation of a surface Ca2+ "receptor". It is unclear whether or not Ca(2+)-induced [Ca2+]i elevation involves the direct gating, by the putative "receptor", of a divalent cation channel. The results show that [Ca2+]i elevation in response to elevated [Ca2+]e comprises a distinct component of Ca2+ influx, the magnitude of which can be decreased and increased, respectively, by depolarising (100 mM-[K+]) and hyperpolarising (1 microM-[valinomycin]) the osteoclast membrane. In addition, activation of the putative Ca2+ "receptor" by elevated [Ca2+]e causes influx of the related divalent cation, magnesium (Mg2+). We suggest that Ca2+ influx induced by Ca2+ "receptor" activation is a major component of the observed [Ca2+]i response.