Optimal ATP Concentration Research Articles

Functional Characterization and Screening of Promiscuous Kinases and Isopentenyl Phosphate Kinases for the Synthesis of DMAPP via a One-Pot Enzymatic Cascade.

Dimethylallyl diphosphate (DMAPP) is a key intermediate metabolite in the synthesis of isoprenoids and is also the prenyl donor for biosynthesizing prenylated flavonoids. However, it is difficult to prepare DMAPP via chemical and enzymatic methods. In this study, three promiscuous kinases from Shigella flexneri (SfPK), Escherichia coli (EcPK), and Saccharomyces cerevisiae (ScPK) and three isopentenyl phosphate kinases from Methanolobus tindarius (MtIPK), Methanothermobacter thermautotrophicus str. Delta H (MthIPK), and Arabidopsis thaliana (AtIPK) were cloned and expressed in Escherichia coli. The enzymatic properties of recombinant enzymes were determined. The Kcat/Km value of SfPK for DMA was 6875 s-1 M-1, which was significantly higher than those of EcPK and ScPK. The Kcat/Km value of MtIPK for DMAP was 402.9 s-1 M-1, which was ~400% of that of MthIPK. SfPK was stable at pH 7.0-9.5 and had a 1 h half-life at 65 °C. MtIPK was stable at pH 6.0-8.5 and had a 1 h half-life at 50 °C. The stability of SfPK and MtIPK was better than that of the other enzymes. Thus, SfPK and MtIPK were chosen to develop a one-pot enzymatic cascade for producing DMAPP from DMA because of their catalytic efficiency and stability. The optimal ratio between SfPK and MtIPK was 1:8. The optimal pH and temperature for the one-pot enzymatic cascade were 7.0 and 35 °C, respectively. The optimal concentrations of ATP and DMA were 10 and 80 mM, respectively. Finally, maximum DMAPP production reached 1.23 mM at 1 h under optimal conditions. Therefore, the enzymatic method described herein for the biosynthesis of DMAPP from DMA can be widely used for the synthesis of isoprenoids and prenylated flavonoids.

P7. Abstract Title: Mapping the Interactome of the Metalloprotease ADAMTS13 in Plasma using In-Vitro BioID

<h3>Background</h3> Von Willebrand Factor (VWF) is a multimeric plasma protein that tethers platelets to sites of vascular damage, and is an important initiator of blood clotting. ADAMTS13 is a metalloprotease that cleaves VWF to regulate its platelet binding activity. Decreased activity of ADAMTS13 leads to larger VWF multimers resulting in pathological blood clots, such as thrombotic thrombocytopenic purpura (TTP) and stroke. Unlike other circulating proteases, ADAMTS13 is secreted as an active enzyme and is resistant to all known natural protease inhibitors. Despite this seemingly unregulated protease activity, VWF is the only known substrate for ADAMTS13. As a result, ADAMTS13 is an enigmatic protease whose role in the cardiovascular system remains poorly understood. <h3>Objective</h3> Identify novel physiologic substrates/ligands for ADAMTS13 in plasma using in-vitro BioID. <h3>Method</h3> BioID is a proximity-dependent biotinylation technique in which the protein of interest (ADAMTS13), is fused with a variant of biotin ligase (BirA*). BirA* activates biotin that can diffuse away and covalently attach to proteins within a 10 nm radius. Therefore, proteins interacting with the bait protein can be labelled with biotin. The biotinylated proteins are isolated using streptavidin pulldown and identified by LC/MS/MS. This technique has successfully been used to identify protein interaction networks in cells. <h3>Results</h3> In preliminary studies, we cloned, expressed, and characterized ADAMTS13-BirA* fusion. The recombinant fusion protein exhibits normal ADAMTS13 proteolytic activity and comparable biotinylation activity relative to BirA* alone. Preliminary LC-MS/MS data from labelling in 100 μL plasma yielded 36 unique proteins. ADAMTS13 was the most abundant protein, consistent with auto-biotinylation of the fusion protein. Other unique proteins labelled were vitronectin, PON1, and actin-B. However, the fusion protein did not label VWF possibly to due to the absence of shear stress (which is known to facilitate VWF cleavage by ADAMTS13) during labelling. Validation studies showed that both the fusion protein and BirA* alone bind to vitronectin. These data suggest that sensitivity and specificity of the assay requires improvement. We showed that ATP has a half-life of ∼4 minutes in plasma, which may limit the sensitivity of the BioID assay. While phosphatase inhibitors such as NaVO4, EDTA, IBMX or NBTI improved ATP half-life, they also blocked the activity of BirA*. Therefore, to improve labelling efficiency, we simply add ATP throughout the labelling experiment to maintain optimal ATP concentrations. These data demonstrate the viability of an in-vitro BioID experiment in human plasma, and optimized reaction conditions will yield novel substrates/ligands for ADAMTS13. <h3>Conclusion</h3> BioID may be a promising discovery tool for identifying novel protease interactions.

On-chip dual enzyme activity assay to investigate regulation of the transamidase and kinase activities of transglutaminase 2

Transglutaminase 2 (TGase2), a multifunctional enzyme exhibiting both transamidase and kinase activity, is involved in a variety of cellular processes and diseases. However, details of the regulation of TGase2 have not been reported due to the lack of a suitable assay to examine both activities on the same platform under near-physiologic conditions. Thus, we developed an on-chip dual enzyme activity assay for TGase2 to simultaneously monitor the transamidase and kinase activities. Reaction mixtures specific for each enzyme activity were applied onto osteopontin arrays, and enzyme activity was monitored by sequential probing with Cy5-strepavidin and Pro-Q Diamond stain. This approach was used to determine the optimal concentrations of ATP, Mg2+, and Ca2+ for dual-activity assays. The optimized assay was then used to investigate regulation of TGase2 transamidase and kinase activities by various cofactors that could potentially affect its conformation. Monothiol- and disulfide-containing compounds differentially regulated TGase2 transamidase and kinase activities. Acetylation of TGase2 activated the kinase activity but had no effect on the transamidase activity. Phosphorylation and dephosphorylation of TGase2 reciprocally regulated the transamidase and kinase activities. The new approach described here is thus useful for screening potential regulators of TGase2 transamidase and kinase and investigating the pathogenesis of TGase2-associated diseases.

Abstract LB-16: Discovery of novel SGK1 inhibitors by a fragment-based approach: Generating patentable, chemically tractable and ligand efficient leads

Abstract Triple negative (ER, PR and HER2/neu negative) breast cancer (TNBC) can have an unfavorable prognosis compared to other breast cancers. TNBC is more difficult to treat since most targeted breast cancer therapies are directed at one of the three receptors. Aggressive combinatorial therapies are usually required and the cytotoxic agents used in these regimens frequently loose effectiveness with time. Serum and glucocorticoid-regulated kinase 1 (SGK1) (a serine/threonine protein kinase), promotes the growth and invasiveness of breast cancer cells. SGK1 is a key protein regulated by the glucocorticoid receptor (GR). In TNBC tumors, high expression/activity in the GR/SGK axis correlates with shorter survival times and resistance to chemotherapy. The small numbers of SGK1 inhibitors reported in the literature have resulted from screening against other kinases (such as CHK1/azaindoles) and thus are not selective and lack patentable chemical diversity. We describe the identification of novel SGK1 inhibitors using a fragment-based library screening approach. The BioBlocks Leap-To-Lead platform contains a library of novel fragment-like compounds that span a range of polarities and sizes without compromising on flexibility. For example, this 275 compound library has an average molecule weight of 160 Da; AlogP, 1.1; hydrogen bond donors, 1.1; hydrogen bond acceptors, 2.2, and rotatable bonds, 0.7 yet still contains 114 unique rings. The library was screened against fully activated human SGK1 in an ADP-Glo assay using physiologically relevant peptide substrate and an optimal ATP concentration to also identify non-classical kinase inhibitor mode binders. The fragment library was screened at 2 mM and hits were profiled in IC50 dose response. From these, several novel chemical matter hit compounds were selected that had ligand efficiency values greater than 0.3 and spanned IC50 values from 75 µM to 2000 µM. In summary, a fragment-based screening approach was used to identify several novel scaffolds that represent both classical and non-classical kinase inhibitor binding modes as hit-to-lead starting points for the optimization of novel, potent and selective SGK1 inhibitors. Citation Format: Gordon Alton, Warren Wade, Laura Lingardo, Ayse Batova, Tom Smart, Chris Buhr, James W. Zapf, Peter Pallai. Discovery of novel SGK1 inhibitors by a fragment-based approach: Generating patentable, chemically tractable and ligand efficient leads. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-16. doi:10.1158/1538-7445.AM2014-LB-16

Micro-colorimetric determination of adenosinetriphosphatase activity in freeze-dried sections of rat diaphragm muscle.

Abstract A technique is described for determination of ATPase activity in 3–30 μg samples dissected from freeze-dried sections of rat diaphragm muscle. Tissue samples are incubated at room temperature with optimal concentrations of ATP (10 mM) and MgSO4 (5 mM) in 0·1 M tris/HCl buffer at pH 7·4. Inorganic phosphate is measured spectrophotometrically. Mean activity for 20 rats was 0·22 ± 0·06 (s.d.) mole-Pi/kg/wet tissue/15 min. Results did not depend on plane of section or section thickness. 5 mM Mg2+, 10 mM Ca2+ and 1 mM 2, 4-dinitrophenol produced maximal ATPase activation and inhibition was obtained with p-chloromercuribenzoic acid (pI503·9) but not chlorpromazine (0·01–0·1 mM). There was no significant evidence of Na+ plus K+ activated ATPase or inhibition with ouabain (5 mM). ATPase activity was uncharacterized and relative contributions of specified muscle ATPase systems were unknown. As an insoluble enzyme system was involved and photomicrographs showed characteristic muscle features in freeze-dried sections, ATPase activity appeared due to enzymic function in situ. Results are discussed in relation to mutual availability of enzymic sites and reagents.

Physiological levels of ATP negatively regulate proteasome function

Intracellular protein degradation by the ubiquitin-proteasome system is ATP dependent, and the optimal ATP concentration to activate proteasome function in vitro is ∼100 μM. Intracellular ATP levels are generally in the low millimolar range, but ATP at a level within this range was shown to inhibit proteasome peptidase activities in vitro. Here, we report new evidence that supports a hypothesis that intracellular ATP at the physiological levels bidirectionally regulates 26S proteasome proteolytic function in the cell. First, we confirmed that ATP exerted bidirectional regulation on the 26S proteasome in vitro, with the optimal ATP concentration (between 50 and 100 μM) stimulating proteasome chymotrypsin-like activities. Second, we found that manipulating intracellular ATP levels also led to bidirectional changes in the levels of proteasome-specific protein substrates in cultured cells. Finally, measures to increase intracellular ATP enhanced, while decreasing intracellular ATP attenuated the ability of proteasome inhibition to induce cell death. These data strongly suggest that endogenous ATP within the physiological concentration range can exert a negative impact on proteasome activities, allowing the cell to rapidly upregulate proteasome activity on ATP reduction under stress conditions.

Mitochondrial shape changes: orchestrating cell pathophysiology

Mitochondria are highly dynamic organelles, the location, size and distribution of which are controlled by a family of proteins that modulate mitochondrial fusion and fission. Recent evidence indicates that mitochondrial morphology is crucial for cell physiology, as changes in mitochondrial shape have been linked to neurodegeneration, calcium signalling, lifespan and cell death. Because immune cells contain few mitochondria, these organelles have been considered to have only a marginal role in this physiological context-which is conversely well characterized from the point of view of signalling. Nevertheless, accumulating evidence shows that mitochondrial dynamics have an impact on the migration and activation of immune cells and on the innate immune response. Here, we discuss the roles of mitochondrial dynamics in cell pathophysiology and consider how studying dynamics in the context of the immune system could increase our knowledge about the role of dynamics in key signalling cascades.

Optimal determination of heart tissue 26S-proteasome activity requires maximal stimulating ATP concentrations

The ubiquitin–proteasome system has been implicated in both cardiac physiology and pathophysiology. Research in this area has been hampered by the lack of a simple, reproducible method to assess 26S-proteasome peptidase activities. The current report demonstrates that one reason for lack of reproducibility is the myriad of ATP concentrations, many of them excessive, which have been used to stimulate peptidase activity. The chymotrypsin-like or caspase-like activities of 26S-proteasome in cardiac tissue isolates were determined using Suc-LLVY-AMC or Z-LLE-AMC, respectively, over a range of ATP concentrations up to 2 mmol/L. The optimal ATP concentration to assess both peptidase activities was found to be in the low micromolar range (from 6 to 100 μmol/L) depending on the cardiac tissue isolate protein (10 to 90 μg protein) contained in the reaction. Increasing ATP beyond the optimal range was inhibitory. In general, chymotrypsin-like and caspase-like activities could be stimulated 2- to 2.5-fold and 1.4- to 1.8-fold, respectively, over basal (ATP, 0 μmol/L), and could be effectively inhibited with lactacystin or Z-Pro-Nle-Asp-CHO, respectively. Based on these observations, an optimized method is presented for ex vivo determination of cardiac 26S-proteasome peptidase activities which was used to confirm inactivation of this complex by myocardial ischemia and reperfusion.

Binding of a Single Rb+ Increases Na+/K+-ATPase, Activating Dephosphorylation without Stoichiometric Occlusion

We used partially purified Na+/K+-ATPase from pig kidney to study dephosphorylation, occlusion, and ATPase activity in the same enzyme preparation and in media of identical composition containing 10 microM ATP and different concentrations of Rb+, used as a K+ congener. The experiments were performed using a rapid-mixing apparatus with a time resolution of 3.5 ms. The main findings were as follows. (i) At sufficiently low Rb+ concentration the initial rate of dephosphorylation was higher than that of occlusion, (ii) as [Rb+] tended to zero the slope of the time course of occlusion but not that of the time course of dephosphorylation approached zero and, (iii) as Rb+ concentration increased, ATPase activity first increased and, after passing through a maximum, tended to a value that was lower than that observed in media without Rb+. None of these results is compatible with the currently held idea that binding of a single Rb+ to the E2P conformer of the ATPase does not modify the rate of dephosphorylation and strongly suggest that a single Rb+ does promote dephosphorylation through a mechanism that is not stoichiometrically coupled to Rb+ occlusion. If this mechanism is included in the currently accepted scheme for ATP hydrolysis by the Na+/K+-ATPase, a reasonable prediction of the experimental results is obtained.

Kinetics and mechanism of ATP-dependent IL-1 beta release from microglial cells.

Endotoxin-dependent release of IL-1 beta from mouse microglial cells is a very inefficient process, as it is slow and leads to accumulation of a modest amount of extracellular cytokine. Furthermore, secreted IL-1 beta is mostly in the procytokine unprocessed form. Addition of extracellular ATP to LPS-primed microglia caused a burst of release of a large amount of processed IL-1 beta. ATP had no effect on the accumulation of intracellular pro-IL-1 beta in the absence of LPS. In LPS-treated cells, ATP slightly increased the synthesis of pro-IL-1 beta. Optimal ATP concentration for IL-1 beta secretion was between 3 and 5 mM, but significant release could be observed at concentrations as low as 1 mM. At all ATP concentrations IL-1 beta release could be inhibited by increasing the extracellular K+ concentration. ATP-dependent IL-1 beta release was also inhibited by 90 and 60% by the caspase inhibitors YVAD and DEVD, respectively. Accordingly, in ATP-stimulated microglia, the p20 proteolytic fragment derived from activation of the IL-1-beta-converting enzyme could be detected by immunoblot analysis. These experiments show that in mouse microglial cells extracellular ATP triggers fast maturation and release of intracellularly accumulated IL-beta by activating the IL-1-beta-converting enzyme/caspase 1.

A Rat RuvB-like Protein, TIP49a, Is a Germ Cell-enriched Novel DNA Helicase

We have isolated a novel nuclear protein with a molecular mass of 49 kDa (TIP49a) from rat liver. The rat TIP49a showed structural resemblance to several bacterial RuvBs and also displayed Walker A and B motifs. We overproduced the recombinant TIP49a in Escherichia coli and purified it to near homogeneity. Biochemical investigations demonstrated that TIP49a possessed ATPase activity that was stimulated by single-stranded DNA but neither by double-stranded DNA nor by any forms of RNA polymers tested. Moreover, a UV cross-linking assay indicated TIP49a specifically interacted with ATP. Interestingly, we found that DNA duplex was unwound by the recombinant TIP49a in the presence of ATP or dATP. Optimal concentrations of ATP and Mg2+ for the helicase activity were 1-2 mM and 0.25-1 mM, respectively. Displacement of the DNA strand occurred in the 3' to 5' direction with respect to the single-stranded DNA flanking the duplex. Western blot analysis revealed that TIP49a was abundantly expressed in testes and moderately in spleen, thymus, and lung. In mouse seminiferous tubules, the protein was restrictively observed in germ lineages from late pachytene spermatocytes to round spermatids. From these observations, we propose that TIP49a is a novel DNA helicase and may play a role in nuclear processes such as recombination and transcription.

Glucose Determination Using a Flow System with Enzyme Reactor and Application to Analysis of Glucose Content in Beverages

Glucose was determined using an apparatus containing an enzyme reactor in a flow line. The enzymes used for glucose assay were hexokinase and glucose-6-phosphate dehydrogenase. NADH formed by enzymatic reactions was fluorometrically detected. The optimal concentrations of ATP and NAD in the carrier were determined. Of the buffers examined for use as a carrier medium, triethanolamine buffer was found to be the most favorable and the optimal pH of this buffer was pH 7.0. Under these optimal conditions, the calibration curve for glucose was linear in the range of 2.0-200μM(r=0.999). Detection limit was 1.7μM(S/N=3).This method was applied to the analysis of glucose in several beverages. Glucose content determined by the present method agreed with that determined by a commercially available test-kit method. The methods for the preparation of immobilized enzymes were examined to retain the activities of enzymes.

The effect of Mg2+, nucleotides and ATPase inhibitors on the uptake of [3H]-cGMP to inside-out vesicles from human erythrocytes.

An ATP-dependent transport system is responsible for the cellular extrusion of cGMP. The objective of the present study was to determine the effect of Mg2+, ATP and other nucleotides (2'-dATP, GTP and ADP), exogenous ATPase modulators (such as metavanadate, ouabain, EGTA, NEM, bafilomycin A1 and oligomycin A) on the cGMP transport. The uptake of [3H]-cGMP (1 microM) at 37 degrees C was studied in inside-out vesicles from human erythrocytes. Magnesium caused a maximal activation between 5 and 10 mM and the optimal ATP concentration was 1.25 mM with K50-values of 0.3-0.5 mM. Among other nucleotides tested, 2'-dATP (K50 of 0.7 mM) was nearly as effective as ATP, whereas cGMP accumulated slowly in the presence of GTP. ADP and metavanadate (P-type ATPase inhibitor) showed to be competitive inhibitors with Ki values of 0.15 mM and 10 microns, respectively. NEM (a sulphydryl agent) reduced the ATP-dependent uptake in a concentration-dependent manner with a Ki value of 10 microM. Ouabain (Na+/K(+)-ATPase inhibitor) had no effect. Bafilomycin A1 (V-type ATPase inhibitor) and oligomycin (F-type ATPase inhibitor) were the most potent inhibitors with Ki values of 0.7 and 1.8 microM, respectively. The present study suggests that the cellular cGMP extrusion is energized by an ATPase with a unique inhibitor profile, which clearly differentiates it from the other major classes of membrane-bound ATPases.

Post-Translational Processing of Murine IL-1: Evidence that ATP-Induced Release of IL-1α and IL-1β Occurs via a Similar Mechanism

AbstractIn response to LPS, peritoneal macrophages produce IL-1, but, for the most part, newly synthesized cytokine molecules remain cell associated. Externalization and proteolytic processing of pro-IL-1β can be initiated by extracellular ATP. In this study, kinetics and inhibitor sensitivity of the stimulus-coupled mechanism were investigated with [35S]methionine-labeled macrophages. Optimal ATP concentrations required to promote cytokine post-translational processing suggest the involvement of a P2Z type of receptor. Proteolysis of pro-IL-1β initiates within 7.5 min of ATP addition; 17-kDa mature IL-1β is observed first intracellularly and subsequently extracellularly. In contrast, ATP-treated cells do not contain 17-kDa IL-1α. Macrophages exposed to ATP continuously or only for a 15-min pulse release IL-1α, IL-1β, and lactate dehydrogenase (LDH). Proteolytic maturation of IL-1β exceeds that of IL-1α in both formats, but pulsed cells process the externalized cytokines more efficiently. Ethacrynic acid and DIDS (4,4′-diisothiocyanato-stilbene-2,2′-disulfonic acid) block ATP-induced proteolysis of pro-IL-1β and prevent release of pro-IL-1α/β and LDH; they do not inhibit ATP-induced K+ (86Rb+) efflux. Ethacrynic acid inhibits release of both forms of IL-1 with a similar concentration dependence; within the arrested cells, procytokines accumulate in a Triton-insoluble fraction. An IL-1β-converting enzyme inhibitor blocks proteolysis of IL-1β, but it does not prevent release of pro-IL-1α, pro-IL-1β, or LDH. These results indicate that ATP stimulates externalization of both IL-1α and IL-1β. The ATP-induced cytokine release mechanism is accompanied by cell death and requires activity of an anion transport inhibitor-sensitive component, but this pathway operates independently of cytokine proteolytic processing.

Purification and characterization of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase from Penicillium chrysogenum.

delta-(L-alpha-Aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) from Penicillium chrysogenum was purified to homogeneity by a combination of (NH4)2SO4 precipitation, protamine sulphate treatment, ion-exchange chromatography, gel filtration and hydrophobic interaction chromatography. The molecular mass of ACVS was estimated with native gradient gel electrophoresis and SDS/PAGE. The native enzyme consisted of a single polymer chain with an estimated molecular mass of 470 kDa. The denatured enzyme had an estimated molecular mass of 440 kDa. The influence of different reaction parameters such as substrates, cofactors and pH on the activity of the purified ACVS was investigated. The Km values for the three precursor substrates L-alpha-aminoadipic acid, L-cysteine and L-valine were determined as 45, 80 and 80 microM respectively, and the optimal assay concentration of ATP was found to be 5 mM (with 20 mM MgCl2). The dimer of the reaction product bis-delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (bisACV) gave feedback inhibition of the purified ACVS; the inhibition parameter KbisACV was determined as 1.4 mM. Furthermore dithiothreitol was shown to inhibit the purified ACVS. From the addition of a glucose pulse to a steady-state glucose-limited continuous culture of P. chrysogenum it was found that there is glucose repression of the synthesis of ACVS and that there must be a constant turnover of ACVS owing to synthesis and degradation.

Characterization of mevalonate metabolism in the sea bass (Dicentrarchus labrax L) liver

The activities of mevalonate kinase, mevalonate 5-phosphate kinase and mevalonate 5-pyrophosphate decarboxylase, were examined in sea bass (Dicentrarchus labrax L) liver. The activities of the three enzymes were studiedin vitro in relation to the influence of protein content, time of incubation, pH, temperature, mevalonate, ATP and Mg(++) concentration. Protein content in the assay medium affected the three enzymes differently. Mevalonate kinase, mevalonate 5-phosphate kinase, and mevalonate 5-pyrophosphate decarboxylase activities were linear up to 0.2, 0.4 and 0.8 mg protein, respectively. With respect to the time course studies, the enzymes also behaved differently. Mevalonate kinase activity increased over forty minutes, reaching a plateau thereafter, while mevalonate 5-phosphate kinase and decarboxylase increased over the entire assay period. All the three enzymes showed a maximum in activity at pH 7.5. The effect of reaction temperature showed that phosphorylation increased to maximum around 35°C for mevalonate kinase and 30°C for mevalonate 5-phosphate kinase while decarboxylation rates remained constant well until 30°C temperature decreasing afterwards. The enzymes behaved differently as a function of mevalonate concentration. Mevalonate 5-phosphate formed was maximal when the initial mevalonate concentration was 272 μM, whereas mevalonate 5-pyrophosphate and CO2 were formed maximally at mevalonate concentrations of 136 μM and 68μM, respectively. Optimal ATP concentration in the medium was 3 mM for decarboxylase and 6 mM for kinases, and Mg(++) requirements varied from 4 mM for decarboxylase to 6 mM for kinases.

Different mechanisms of Ca2(+)-handling following nicotinic acetylcholine receptor stimulation, P2U-purinoceptor stimulation and K(+)-induced depolarization in C2C12 myotubes.

1. The increase in intracellular CA2+ on nicotinic acetylcholine receptor (nAChR) stimulation, P2U-purinoceptor stimulation and K(+)-induced depolarization was investigated in mouse C2C12 myotubes by use of fura-2 fluorescence to characterize the intracellular organisation of Ca2+ releasing stores and Ca(2+)-entry process. 2. Stimulation of nAChRs with carbachol induced a rapid rise in internal Ca2+ (EC50 = 0.85 +/- 0.09 microM), followed by a sustained phase. The Ca2+ response evoked by carbachol (10 microM) was completely blocked by the nAChR antagonist, pancuronium (3 microM), but was not affected by the muscarinic antagonist, atropine (3 microM), or under conditions when Ca2+ entry was blocked by La3+ (50 microM) or diltiazem (10 microM). Addition of pancuronium (3 microM) during the sustained phase of the carbachol-evoked response did not affect this phase. 3. Stimulation of P2U purinoceptors with ATP (1 mM) induced a somewhat higher biphasic Ca2+ response (EC50 of the rapid phase: 8.72 +/- 0.08 microM) than with carbachol. Pretreatment with La3+ abolished the sustained phase of the ATP-induced Ca2+ response, while the response was unaffected by diltiazem or pancuronium. 4. Stimulation of the cells with high K+ (60 mM), producing the same depolarization as with carbachol (10 microM), induced a rapid monophasic Ca2+ response, insensitive to diltiazem, pancuronium or La3+. 5. Under Ca(2+)-free conditions, the sustained phase of the carbachol- and ATP-evoked responses were abolished. Pre-emptying of depolarization-sensitive stores by high K+ under Ca(2+)-free conditions did not affect the carbachol- or ATP-evoked Ca2+ mobilization and vice versa. Preincubation of the cells with ATP in the absence of extracellular Ca2+ decreased the amplitude of the subsequent carbachol-induced Ca2+ response to 11%, while in the reverse procedure the ATP-induced response was decreased to 65%. Ca2+ mobilization evoked by simultaneous addition of optimal concentrations of carbachol and ATP was increased compared to levels obtained with either agonist. 6. Preincubation with high K+ under normal conditions abolished the sustained phase of the ATP-evoked Ca2+ response. The carbachol response consisted only of the sustained phase in the presence of high K+. 7. The carbachol-induced Ca2+ response was completely abolished under low Na+/Ca(2+)-free conditions, while under low Na+ conditions only a sustained Ca2+ response was observed. The ATP- and K(+)-induced responses were changed compared to Ca(2+)-free conditions. 8. ATP (300 microM) induced the formation of Ins(1,4,5)P3 under Ca(2+)-free conditions with a comparable time course to that found for the rise in internal Ca2+. In contrast to ATP, carbachol (10 microM) did not affect Ins(1,4,5)P3 levels under Ca(2+)-free conditions. 9. It is concluded that the Ca2+ release from discrete stores of C2C12 myotubes is induced by stimulation of nAChRs, P2U-purinoceptors and by high K+. Only the P2U-purinoceptor and nAChR activated stores show considerable overlap in releasable Ca2+. Sustained Ca(2+)-entry is activated by stimulation of nAChRs and P2U-purinoceptors via separate ion-channels, which are different from the skeletal muscle nAChR-coupled cation-channel.

Agonist‐Specific Calcium Signaling and Phosphoinositide Hydrolysis in Human SK‐N‐MCIXC Neuroepithelioma Cells

Fura-2 digital imaging microfluorimetry was used to evaluate the Ca2+ signals generated in single clonal human neuroepithelioma cells (SK-N-MCIXC) in response to agonists that stimulate phosphoinositide hydrolysis. Addition of optimal concentrations of either endothelin-1 (ET-1), ATP, oxotremorine-M (Oxo-M), or norepinephrine (NE) all resulted in an increase in the concentration of cytosolic calcium (Ca2+i) but of different magnitudes (ET-1 = ATP > Oxo-M > NE). The Ca2+ signals elicited by the individual agonists also differed from each other in terms of their latency of onset, rate of rise and decay, and prevalence of a sustained phase of Ca2+ influx. The Ca2+ signals that occurred in response to ATP had a shorter latency and more rapid rates of rise and decay than those observed for the other three agonists. Furthermore, a sustained plateau phase of the Ca2+ signal, which was characteristic of the response to Oxo-M, was observed in < 40% of cells stimulated with ET-1 and absent from Ca2+ signals elicited after NE addition. Removal of extracellular Ca2+ enhanced the rate of decay of Ca2+ signals generated by ATP, ET-1, or Oxo-M and, when evident, abolished the sustained phase of Ca2+ influx. In the absence of extracellular Ca2+, NE elicited asynchronous multiple Ca2+ transients. In either the absence or presence of extracellular Ca2+-, > 94% of cells responded to ET-1 or ATP, whereas corresponding values for Oxo-M and NE were approximately 74 and approximately 48%.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of the phospholipase C pathway by ATP is mediated exclusively through nucleotide type P2-purinoceptors in C2C12 myotubes.

1. The presence of a nucleotide receptor and a discrete ATP-sensitive receptor on C2C12 myotubes has been shown by electrophysiological experiments. In this study, the ATP-sensitive receptors of C2C12 myotubes were further characterized by measuring the formation of inositol(1,4,5)trisphosphate (Ins(1,4,5)P3) and internal Ca2+. 2. The nucleotides ATP and UTP caused a concentration-dependent increase in Ins(1,4,5)P3 content with comparable time courses (EC50: ATP 33 +/- 2 microM, UTP 80 +/- 4 microM). ADP was less effective in increasing Ins(1,4,5)P3 content of the cells, while selective agonists for P1-, P2X- and P2Y-purinoceptors, adenosine, alpha,beta-methylene ATP and 2-methylthio ATP, appeared to be ineffective. 3. Under Ca(2+)-free conditions, the basal level of Ins(1,4,5)P3 was lower than in the presence of Ca2+, and the ATP- and UTP-induced formation of Ins(1,4,5)P3 was diminished. 4. The Ins(1,4,5)P3 formation induced by optimal ATP and UTP concentrations was not additive. ATP- and UTP-induced Ins(1,4,5)P3 formation showed cross-desensitization, whereas cross-desensitization was absent in responses elicited by one of the nucleotides and bradykinin. 5. The change in Ins(1,4,5)P3 content induced by effective nucleotides was inhibited by suramin. Schild plots for suramin inhibition of Ins(1,4,5)P3 formation in ATP- and UTP-stimulated myotubes showed slopes greater than unity (1.63 +/- 0.09 and 1.37 +/- 0.11, respectively). Apparent pA2 values were 4.50 +/- 0.48 and 4.41 +/- 0.63 for ATP and UTP, respectively. 6. Stimulation of the cells with ATP or UTP induced a rapid increase in intracellular Ca2+, followed by a slow decline to basal levels. Ca2+ responses reached lower maximal values and did not show the slow phase in the absence of extracellular Ca2+. The ATP and UTP-evoked increase in intracellular Ca2+ was not additive and showed cross-desensitization. Cross-desensitization was absent in myotubes stimulated with one of the nucleotides and bradykinin.7. These results show that ATP- and UTP-induced formation of Ins(1,4,5)P3, Ca2+ release from internal stores and Ca2+-influx from the extracellular space are mediated exclusively via the nucleotide type P2-purinoceptor in mouse C2C12 myotubes.

Characterization of ATP-dependent Ca2+ transport in the basolateral membrane vesicles from proximal and distal tubules of the rabbit kidney

Basolateral membrane vesicles were prepared from purified proximal and distal tubules of the rabbit kidney. The properties of the ATP-dependent Ca2+ transport were investigated. In both membranes, there was a high affinity, ATP-dependent Ca2+ transport system (Km = 0.1 microM). The optimal concentration of Mg2+ was 0.5 mM and the optimal concentration of ATP was 1 mM. The nucleotide specificity and pH dependence of the Ca2+ transport in both membranes were similar. In basolateral membrane vesicles, calmodulin had no effect on Ca2+ transport. However, in basolateral membrane vesicles depleted of calmodulin, exogenous calmodulin increased the Ca2+ transport by increasing maximal velocity. There were no major differences in the properties of the ATP-dependent Ca2+ transport system in these two membranes. These findings are discussed in relation to why parathyroid hormone differentially modulates Ca2+ transport in these two segments of the nephron.