Helicobacter pylori lipopolysaccharide enhances the expression of NADPH oxidase components in cultured guinea pig gastric mucosal cells.

Recently, we showed that cultured guinea pig gastric pit cells possess a phagocyte NADPH oxidase-like activity, which was up-regulated by Helicobacter pylori lipopolysaccharide. We demonstrate here that these cells express all of the phagocyte NADPH oxidase components (gp91-, p22-, p67-, p47-, and p40-phoxes). Treatment with lipopolysaccharide increased the expression of gp91-, p22-, and p67-phoxes, but not that of p47- and p40-phoxes. Intriguingly, the p67-phox expression consistently correlated with up-regulation of superoxide anion-producing ability. Thus, the gastric pit cell NADPH oxidase may play an important role in regulation of the inflammatory response associated with H. pylori infection.     

Role of components of the phagocytic NADPH oxidase in oxygen sensing.

It has been hypothesized that O(2) sensing in type I cells of the carotid body and erythropoietin (EPO)-producing cells of the kidney involves protein components identical to the NADPH oxidase system responsible for the respiratory burst of phagocytes. In the present study, we evaluated O(2) sensing in mice with null mutant genotypes for two components of the phagocytic oxidase. Whole body plethysmography was used to study unanesthetized, unrestrained mice. When exposed to an acute hypoxic stimulus, gp91(phox)-null mutant and wild-type mice increased their minute ventilation by similar amounts. In contrast, p47(phox)-null mutant mice demonstrated increases in minute ventilation in response to hypoxia that exceeded that of their wild-type counterparts: 98.0 +/- 18.0 vs. 20.0 +/- 13.0% (n = 11, P = 0.003). In vitro recordings of carotid sinus nerve (CSN) activity demonstrated that resting (basal) neural activity was marginally elevated in p47(phox)-null mutant mice. With hypoxic challenge, mean CSN discharge was 1.5-fold greater in p47(phox)-null mutant than in wild-type mice: 109.61 +/- 13.29 vs. 72.54 +/- 7.65 impulses/s (n = 8 and 7, respectively, P = 0.026). Consequently, the hypoxia-evoked CSN discharge (stimulus-basal) was approximately 58% larger in p47(phox)-null mutant mice. Quantities of EPO mRNA in kidney were similar in gp91(phox)- and p47(phox)-null mutant mice and their respective wild-type controls exposed to hypobaric hypoxia for 72 h. These findings confirm the previous observation that absence of the gp91(phox) component of the phagocytic NADPH oxidase does not alter the O(2)-sensing mechanism of the carotid body. However, absence of the p47(phox) component significantly potentiates ventilatory and chemoreceptor responses to hypoxia. O(2) sensing in EPO-producing cells of the kidney appears to be independent of the gp91(phox) and p47(phox) components of the phagocytic NADPH oxidase.     

Induction of the respiratory burst in HL-60 cells. Correlation of function and protein expression

Differentiation of myeloid cells is associated with the gradual acquisition of functional capacity to produce a respiratory burst. In our study HL-60 cells were differentiated to the monocyte phenotype with IFN-gamma or 1,25-dihydroxyvitamin D3, or to the neutrophil phenotype with retinoic acid or DMSO to compare the time-course of expression of membrane and cytosolic oxidase components, and to correlate this with the appearance of a functional oxidase. Over a 6-day period of induction the rank order of the ability of these agents to induce expression of PMA-stimulated superoxide production was: IFN-gamma greater than 1,25(OH)2D3 greater than retinoic acid greater than DMSO. Immunoblot analysis of HL-60 membranes and cytosol was used to assess the amount of specific phagocyte oxidase factors (91 and 22 kDa subunits of membrane cytochrome b558 (gp91 and p22), and 47 and 67 kDa cytosol oxidase factors (p47 and p67)). HL-60 cell membranes or cytosol were tested in a cell-free assay of superoxide production by mixing with normal neutrophil cytosol or membranes, respectively. p47 was first detected at 16 h of differentiation, increasing similarly thereafter with all induction regimens and reaching a maximum by 3 to 4 days. The earliest detection of p67 varied from 2 to 6 days depending on the inducing agent and appeared to be the limiting cytosol component. Small amounts of both subunits of cytochrome b558 were detected in uninduced HL-60 membranes, but were sufficient to support substantial superoxide production when combined with normal neutrophil cytosol. Both cytochrome b558 subunit proteins and membrane oxidase activity increased during differentiation in parallel. We conclude that membrane and cytosol components of the NADPH oxidase complex appear at different times and increase differently during HL-60 differentiation. The production of p67 is the major factor limiting the respiratory burst during HL-60 differentiation.     

Mapping of functional domains in the p22(phox) subunit of flavocytochrome b(559) participating in the assembly of the NADPH oxidase complex by "peptide walking".

The superoxide-generating NADPH oxidase complex of phagocytes consists of a membranal heterodimeric flavocytochrome (cytochrome b(559)), composed of gp91(phox) and p22(phox) subunits, and four cytosolic proteins, p47(phox), p67(phox), p40(phox), and the small GTPase Rac (1 or 2). All redox stations involved in electron transport from NADPH to oxygen are located in gp91(phox). NADPH oxidase activation is the consequence of assembly of cytochrome b(559) with cytosolic proteins, a process reproducible in a cell-free system, consisting of phagocyte membranes, and recombinant cytosolic components, activated by an anionic amphiphile. p22(phox) is believed to act as a linker between the cytosolic components and gp91(phox). We applied "peptide walking" to mapping of domains in p22(phox) participating in NADPH oxidase assembly. Ninety one synthetic overlapping pentadecapeptides, spanning the p22(phox) sequence, were tested for the ability to inhibit NADPH oxidase activation in the cell-free system and to bind individual cytosolic NADPH oxidase components. We conclude the following. 1) The p22(phox) subunit of cytochrome b(559) serves as an anchor for both p47(phox) and p67(phox). 2) p47(phox) binds not only to the proline-rich region, located at residues 151-160 in the cytosolic C terminus of p22(phox), but also to a domain (residues 51-63) located on a loop exposed to the cytosol. 3) p67(phox) shares with p47(phox) the ability to bind to the proline-rich region (residues 151-160) and also binds to two additional domains, in the cytosolic loop (residues 81-91) and at the start of the cytosolic tail (residues 111-115). 4) The binding affinity of p67(phox) for p22(phox) peptides is lower than that of p47(phox). 5) Binding of both p47(phox) and p67(phox) to proline-rich p22(phox) peptides occurs in the absence of an anionic amphiphile. A revised membrane topology model of p22(phox) is proposed, the core of which is the presence of a functionally important cytosolic loop (residues 51-91).     

Regulation of growth and apoptosis of cultured guinea pig gastric mucosal cells by mitogenic oxidase 1

We previously reported that primary cultures of guinea pig gastric pit cells expressed all of the phagocyte NADPH oxidase components (gp91-, p22-, p67-, p47-, and p40-phox) and could spontaneously release superoxide anion (O(2)(-)). We demonstrate here that pit cells express a nonphagocyte-specific gp91-phox homolog (Mox1) but not gp91-phox. Inclusion of catalase significantly inhibited [(3)H]thymidine uptake during the initial 2 days of culture. Pit cells, matured on day 2, slowly underwent spontaneous apoptosis. Scavenging O(2)(-) and related oxidants by superoxide dismutase plus catalase or N-acetyl cysteine (NAC) and inhibiting Mox1 oxidase by diphenylene iodonium activated caspase 3-like proteases and markedly enhanced chromatin condensation and DNA fragmentation. This accelerated apoptosis was completely blocked by a caspase inhibitor, z-Val-Ala-Asp-CH(2)F. Mox1-derived reactive oxygen intermediates constitutively activated nuclear factor-kappaB, and inhibition of this activity by nuclear factor-kappaB decoy oligodeoxynucleotide accelerated their spontaneous apoptosis. These results suggest that O(2)(-) produced by the pit cell Mox1 oxidase may play a crucial role in the regulation of their spontaneous apoptosis as well as cell proliferation.     

Reconstitution of chemotactic peptide-induced nicotinamide adenine dinucleotide phosphate (reduced) oxidase activation in transgenic COS-phox cells

A whole-cell-based reconstitution system was developed to study the signaling mechanisms underlying chemoattractant-induced activation of NADPH oxidase. This system takes advantage of the lack of formyl peptide receptor-mediated response in COS-phox cells expressing gp91(phox), p22(phox), p67(phox), and p47(phox), which respond to phorbol ester and arachidonic acid with O()(2) production. By exogenous expression of signaling molecules enriched in neutrophils, we have identified several critical components for fMLP-induced NADPH oxidase activation. Expression of PKCdelta, but not PKCalpha, -betaII, and -zeta, is necessary for the COS-phox cells to respond to fMLP. A role of PKCdelta in neutrophil NADPH oxidase was confirmed based on the ability of fMLP to induce PKCdelta translocation and the sensitivity of fMLP-induced O()(2) production to rottlerin, a PKCdelta-selective inhibitor. Optimal reconstitution also requires phospholipase C-beta2 and PI3K-gamma. We found that formyl peptide receptor could use the endogenous Rac1 as well as exogenous Rac1 and Rac2 for NADPH oxidase activation. Exogenous expression of p40(phox) potentiated fMLP-induced O()(2) production and raised the level of O()(2) in unstimulated cells. Collectively, these results provide first direct evidence for reconstituting fMLP-induced O()(2) production in a nonhemopoietic cell line, and demonstrate the requirement of multiple signaling components for optimal activation of NADPH oxidase by a chemoattractant.     

Evaluation of the process for superoxide production by NADPH oxidase in human neutrophils: evidence for cytoplasmic origin of superoxide

We present an up-to-date insight into the function of NADPH oxidase in human neutrophils, the signalling pathways involved in activation of this enzyme and the process of association of its components with the cytoskeleton. We also discuss the functional implications of morphological studies revealing localization of the sites of NADPH oxidase activity. An original model of the process of superoxide (O2*-) production in human neutrophils is shown. Organization of NADPH oxidase is associated with several components. Upon stimulation, tri-phox cytosolic components of NADPH oxidase (p40-phox, p47-phox and p67-phox) bind to actin filaments. This process involves other actin-binding proteins, such as cofilin and coronin. Activated protein kinase C, translocated from the plasma membrane, phosphorylates cytosolic components at a scaffold of cytoskeleton. Subsequently, p40-phox, responsible for maintaining the resting state of NADPH oxidase, is separated from other two cytosolic phox proteins following an attachment of the active form of small GTP-binding protein Rac to p67-phox. Cytosolic duo-phox proteins (p47-phox and p67-phox) conjugate with membrane components (gp91-phox, p22-phox and Rapla) of NADPH oxidase residing within membranes of intracellular compartments. This chain of events triggers production of O2*-. Then, oxidant-producing intracellular compartments associate with the plasma membrane. Eventually, intracellularly produced O2*- is released to the extracellular environment through the orifice formed by fusion of oxidant-producing compartments with the plasma membrane. Intracellular movement of the oxidant-producing compartments may be regulated by myosin light chain kinase. The review emphasizes that functional assembly of NADPH oxidase and, therefore, generation of O2*- is accomplished essentially within the intracellular compartments. Upon neutrophil stimulation, intracellularly generated O2*- is transported to the plasma membrane to be released and to ensure host defense against infection.     

Statistical and mutational analysis of chronic granulomatous disease in Japan with special reference to gp91-phox and p22-phox deficiency

Chronic granulomatous disease (CGD) is a group of inherited disorders of host defense caused by a mutation in any of the four components of phagocyte NADPH oxidase, namely gp91-, p22-, p47-, and p67-phox. We have made a precise statistical analysis of 229 registered patients from 195 families in Japan and mutation analysis of 28 and 5 independent patients, respectively, with gp91- and p22-phox deficiency. The gp91- and p22-phox proteins form the membrane cytochrome b558, which plays important roles in the assembly of the active oxidase and electron-transfer reaction, and the lesions in either subunit account for more than 80% of cases. The ratio of male to female patients was 6.6/1, the incidence was calculated to be about 1 out of 220,000 birth, and the life expectancy of the patients born in the 1970s was estimated to be 25-30 years old. For the X-linked gp91-phox deficiency, we found five missense and nine nonsense mutations, seven deletions, three insertions, and four splice site mutations, which included the following novel mutations: four missense, five nonsense, six deletions, one insertion, and two splice site abnormalities. With regard to p22-phox deficiency, two homozygous nonsense mutations and one homozygous deletion, a missense mutation together with a splice site mutation, and two different missense mutations were found. These mutations have not been reported before. Based on the present and reported data from Japan, we discuss the molecular defects of the disease and the difference in statistics between western countries and Japan.     

Expression of NADPH oxidase in human pancreatic islets

AIMS: NADPH oxidase (NOX) is a known source of superoxide anions in phagocytic and non-phagocytic cells. In this study, the presence of this enzyme in human pancreatic islets and the importance of NADPH oxidase in human β-cell function were investigated. MAIN METHODS AND KEY FINDINGS: In isolated human pancreatic islets, the expression of NADPH oxidase components was evidenced by real-time PCR (p22(PHOX), p47(PHOX) and p67(PHOX)), Western blotting (p47(PHOX) and p67(PHOX)) and immunohistochemistry (p47(PHOX), p67(PHOX) and gp91(PHOX)). Immunohistochemistry experiments showed co-localization of p47(PHOX), p67(PHOX) and gp91(PHOX) (isoform 2 of NADPH oxidase-NOX2) with insulin secreting cells. Inhibition of NADPH oxidase activity impaired glucose metabolism and glucose-stimulated insulin secretion. SIGNIFICANCE: These findings demonstrate the presence of the main intrinsic components of NADPH oxidase comprising the NOX2 isoform in human pancreatic islets, whose activity also contributes to human β-cell function.     

New insights into the membrane topology of the phagocyte NADPH oxidase: characterization of an anti-gp91-phox conformational monoclonal antibody

Cytochrome b(558) is the catalytic core of the phagocyte NADPH oxidase that mediates the production of bactericidal reactive oxygen species. Cytochrome b(558) is formed by two subunits gp91-phox and p22-phox (1/1), non-covalently associated. Its activation depends on the interaction with cytosolic regulatory proteins (p67-phox, p47-phox, p40-phox and Rac) leading to an electron transfer from NADPH to molecular oxygen and to the release of superoxide anions. Several studies have suggested that the activation process was linked to a change in cytochrome b(558) conformation. Recently, we confirmed this hypothesis by isolating cytochrome b(558) in a constitutively active form. To characterize active and inactive cytochrome b(558) conformations, we produced four novel monoclonal antibodies (7A2, 13B6, 15B12 and 8G11) raised against a mixture of cytochrome b(558) purified from both resting and stimulated neutrophils. The four antibodies labeled gp91-phox and bound to both native and denatured cytochrome b(558). Interestingly, they were specific of extracellular domains of the protein. Phage display mapping combined to the study of recombinant gp91-phox truncated forms allowed the identification of epitope regions. These antibodies were then employed to investigate the NADPH oxidase activation process. In particular, they were shown to inhibit almost completely the NADPH oxidase activity reconstituted in vitro with membrane and cytosol. Moreover, flow cytometry analysis and confocal microscopy performed on stimulated neutrophils pointed out the capacity of the monoclonal antibody 13B6 to bind preferentially to the active form of cytochrome b(558). All these data suggested that the four novel antibodies are potentially powerful tools to detect the expression of cytochrome b(558) in intact cells and to analyze its membrane topology. Moreover, the antibody 13B6 may be conformationally sensitive and used as a probe for identifying the active NADPH oxidase complex in vivo.     

Deletion mutagenesis of p22phox subunit of flavocytochrome b558: identification of regions critical for gp91phox maturation and NADPH oxidase activity

The heterodimeric flavocytochrome b558, comprised of the two integral membrane proteins p22phox and gp91phox, mediates the transfer of electrons from NADPH to molecular oxygen in the phagocyte NADPH oxidase to generate the superoxide precursor of microbicidal oxidants. This study uses deletion mutagenesis to identify regions of p22phox required for maturation of gp91phox and for NADPH oxidase activity. N-terminal, C-terminal, or internal deletions of human p22phox were generated and expressed in Chinese hamster ovary cells with transgenes for gp91phox and two other NADPH oxidase subunits, p47phox, and p67phox. The results demonstrate that p22phox-dependent maturation of gp91phox carbohydrate, cell surface expression of gp91phox, and the enzymatic function of flavocytochrome b558 are closely correlated. Whereas the 5 N-terminal and 25 C-terminal amino acids are dispensable for these functions, the N-terminal 11 amino acids of p22phox are required, as is a hydrophilic region between amino acids 65 and 90. Upon deletion of 54 residues at the C terminus of p22phox (amino acids 142-195), maturation and cell surface expression of gp91phox was still preserved, although NADPH oxidase activity was absent, as expected, due to removal of a proline-rich domain between amino acids 151-160 that is required for recruitment of p47phox. Antibody binding studies indicate that the extreme N terminus of p22phox is inaccessible in the absence of cell permeabilization, supporting a model in which both the N- and C-terminal domains of p22phox extend into the cytoplasm, anchored by two membrane-embedded regions.     

Cutting edge: infection by the agent of human granulocytic ehrlichiosis prevents the respiratory burst by down-regulating gp91phox

The agent of human granulocytic ehrlichiosis (HGE) is an emerging tick-borne pathogen that resides in neutrophils and can be cultured in a promyelocytic (HL-60) cell line. In response to microbes, polymorphonuclear leukocytes normally activate the NADPH oxidase enzyme complex and generate superoxide anion (O2-). However, HL-60 cells infected with HGE bacteria did not produce O2- upon activation with PMA. RT-PCR demonstrated that HGE organisms inhibited mRNA expression of a single component of NADPH oxidase, gp91phox, and FACS analysis showed that plasma membrane-associated gp91phox protein was reduced on the infected cells. Infection with HGE organisms also decreased gp91phox mRNA levels in splenic neutrophils in a murine model of HGE, demonstrating this phenomenon in vivo. Therefore, HGE bacteria repress the respiratory burst by down-regulating gp91phox, the first direct inhibition of NADPH oxidase by a pathogen.     

Evaluation of the process for superoxide production by NADPH oxidase in human neutrophils: evidence for cytoplasmic origin of superoxide

Abstract

We present an up-to-date insight into the function of NADPH oxidase in human neutrophils, the signalling pathways involved in activation of this enzyme and the process of association of its components with the cytoskeleton. We also discuss the functional implications of morphological studies revealing localization of the sites of NADPH oxidase activity. An original model of the process of superoxide (O2) production in human neutrophils is shown. Organization of NADPH oxidase is associated with several components. Upon stimulation, tri-phox cytosolic components of NADPH oxidase (p40-phox, p47-phox and p67-phox) bind to actin filaments. This process involves other actin-binding proteins, such as cofilin and coronin. Activated protein kinase C, translocated from the plasma membrane, phosphorylates cytosolic components at a scaffold of cytoskeleton. Subsequently, p40-phox, responsible for maintaining the resting state of NADPH oxidase, is separated from other two cytosolic phox proteins following an attachment of the active form of small GTP-binding protein Rac to p67-phox. Cytosolic duo-phox proteins (p47-phox and p67-phox) conjugate with membrane components (gp91-phox, p22-phox and Rap1a) of NADPH oxidase residing within membranes of intracellular compartments. This chain of events triggers production of O2. Then, oxidant-producing intracellular compartments associate with the plasma membrane. Eventually, intracellularly produced O2 is released to the extracellular environment through the orifice formed by fusion of oxidant-producing compartments with the plasma membrane. Intracellular movement of the oxidant-producing compartments may be regulated by myosin light chain kinase. The review emphasizes that functional assembly of NADPH oxidase and, therefore, generation of O2 is accomplished essentially within the intracellular compartments. Upon neutrophil stimulation, intracellularly generated O2 is transported to the plasma membrane to be released and to ensure host defense against infection.     

Adventitial delivery of dominant-negative p67phox attenuates neointimal hyperplasia of the rat carotid artery

Several essential components of NADPH oxidase, including p22phox, gp91phox (nox2) and its homologs nox1 and nox4, p47phox, p67phox, and rac1, are present in the vasculature. We previously reported that p67phox is essential for adventitial fibroblast NADPH oxidase O2- production. Thus we postulated that inhibition of adventitial p67phox activity would attenuate angioplasty-induced hyperplasia. To test this hypothesis, we treated the adventitia of carotid arteries with a control adenovirus (Ad-control), a virus expressing dominant-negative p67phox (Ad-p67dn), or a virus expressing a competitive peptide (gp91ds) targeting the p47phox-gp91phox interaction (Ad-gp91ds). Common carotid arteries (CCAs) from male Sprague-Dawley rats were transfected with Ad-control, Ad-p67dn, or Ad-gp91ds in pluronic gel. After 2 days, a 2-F (Fogarty) catheter was used to injure CCAs in vivo. After 14 days, CCAs were perfusion-fixed and analyzed. In 13 experiments, digital morphometry suggested a reduction of neointimal hyperplasia with Ad-p67dn compared with Ad-control; however, the reduction did not reach statistical significance (P = 0.058). In contrast, a significant reduction was achieved with Ad-gp91ds (P = 0.006). No changes in medial area or remodeling were observed with either treatment. Moreover, adventitial fibroblast proliferation in vitro was inhibited by Ad-gp91ds but not by Ad-p67dn, despite confirmation that Ad-p67dn inhibits NADPH oxidase in fibroblasts. These data appear to suggest that a multicomponent vascular NADPH oxidase plays a role in neointimal hyperplasia. However, inhibition of p47phox may be more effective than inhibition of p67phox at attenuating neointimal growth.     

翘嘴鳜NADPH氧化酶2个催化亚基cDNA的克隆和表达特征

吞噬细胞NADPH氧化酶能生成用于清除病原微生物的活性氧(reactive oxygen species, ROS),在机体的防御体系中起着非常重要的作用.本文利用RT-PCR结合RACE-PCR的方法,克隆到翘嘴鳜NADPH氧化酶的催化亚基gp91phox和p22phox的cDNA全长.并研究两者在正常的翘嘴鳜和注射了柱状黄杆菌灭活菌苗（FKG4）的翘嘴鳜组织中的表达模式.结果表明,gp91phox基因cDNA序列全长2 037 nt,开放阅读框长度为1 698 nt,翻译成565个氨基酸;p22phox 基因cDNA序列全长1 296 nt,开放阅读框561 nt,翻译成186个氨基酸.将这2个亚基推导的氨基酸序列与人的对应亚基相比,相似性分别为68.7%和60.8%,且具有相似的结构域和功能域,说明翘嘴鳜与人的NADPH氧化酶具有相似的功能活性.半定量PCR分析显示,在翘嘴鳜血液、脑、心脏、肾、肝、脾、胸腺等11种组织中均能检测到gp91phox和p22phox的基因表达.经FKG4免疫后,gp91phox在翘嘴鳜血液、头肾和脾3种组织中的表达量显著上升,p22phox在头肾和脾2种组织中的表达量显著上升.由此推断,NADPH氧化酶可能参与了机体的抗菌免疫应答.     

Superoxide Production by Plant Homologues of the gp91phox NADPH Oxidase. Modulation of Activity by Calcium and by Tobacco Mosaic Virus Infection

Genes encoding homologs of the gp91(phox) subunit of the plasma membrane NADPH oxidase complex have been identified in plants and are hypothesized to be a source of reactive oxygen species during defense responses. However, the direct involvement of the gene products in superoxide (O(2)(-)) production has yet to be shown. A novel activity gel assay based on protein fractionation in native or sodium dodecyl sulfate (SDS)-denaturing polyacrylamide gels was developed. In native polyacrylamide gel electrophoresis, one or two major O(2)(-)-producing formazan bands were detected in tomato (Lycopersicum esculentum Mill. cv Moneymaker) and tobacco (Nicotiana tabacum var. Samsun, NN) plasma membranes, respectively. Denaturing fractionation of tomato and tobacco plasma membrane in SDS-polyacrylamide gel electrophoresis, followed by regeneration of the in-gel activity, revealed NADPH-dependent O(2)(-)-producing formazan bands of 106-, 103-, and 80- to 75-kD molecular masses. The SDS and native activity bands were dependent on NADPH and completely inhibited by diphenylene iodonium or CuZn- O(2)(-) dismutase, indicating that the formazan precipitates were due to reduction by O(2)(-) radicals catalyzed by an NADPH-dependent flavin containing enzyme. The source of the plasma membrane activity bands was confirmed by their cross-reaction with antibody prepared from the C terminus of the tomato gp91(phox) homolog. Membrane extracts as well as the in-gel NADPH oxidase activities were stimulated in the presence of Ca(2+). In addition, the relative activity of the gp91(phox) homolog was enhanced in the plasma membrane of tobacco mosaic virus-infected leaves. Thus, in contrast to the mammalian gp91(phox), the plant homolog can produce O(2)(-) in the absence of additional cytosolic components and is stimulated directly by Ca(2+).