ATP Peaks Research Articles

On the molecular mechanism of excitation–transcription coupling in skeletal muscle

An important question in neuromuscular biology is how skeletal muscle cells decipher the stimulation pattern coming from motoneurons to define their phenotype-activating transcriptional changes in a process named excitation-transcription coupling. We have shown in adult muscle fibers that 20 Hz electrical stimulation (ES) activates a signaling cascade that starts with Cav1.1 activation, ATP release trough pannexin-1 channel, activation of purinergic receptors, and IP3-dependent Ca2+ signals inducing transcriptional changes related to muscle plasticity from fast to slow phenotype. Extracellular addition of 30 µM ATP mimics transcriptional changes induced by ES at 20 Hz. ATP release occurs in two peaks, the first around 15 s after ES and a second around 300 s after ES. In the present work, we used apyrase to hydrolyze ATP 60 s after ES, maintaining the first peak and eliminating the second peak. In this condition, transcriptional changes were abolished, indicating that the second peak is the one crucial to activate transcription. Additionally, we observed a small depolarization of fibers after ES. The addition of 30 to 100 µM external ATP also induced depolarization of muscle fibers. This depolarization was unable to activate contraction but was able to induce transcriptional changes induced by 20 Hz ES. These changes were completely inhibited by the IP3R blocker xestospongin B, suggesting that IP3-dependent events are triggered at these membrane depolarization values. Moreover, transcriptional changes induced by addition of 30 µM extracellular ATP was blocked by incubation of fibers with 25 µM Nifedipine. These results suggest that the second ATP peak observed after 20 Hz ES is responsible for transcriptional activation by inducing small depolarizations of fiber membranes that are also sensed by Cav1.1. Finally, we show evidence that downstream of purinergic receptors, PKC is activated, likely causing phosphorylation of ClC-1 chloride channels, possibly responsible for depolarization after 20 Hz.

Stability-indicating LC method for the simultaneous determination of methyl paraben, propyl paraben, butylated hydroxytoluene and alpha-tocopherol contents in marijuana capsules

The current study is a simple liquid chromatographic method developed and validated for the simultaneous determination of methyl paraben, propyl paraben, butylated hydroxytoluene and alpha-tocopherol in marijuana (delta-8-tetra hydro cannabinol (or) dronabinol) soft gelatin capsules by using high-performance liquid chromatographic technique. The chromatographic separation was achieved on a Waters Xterra RP18 (150 × 4.6)-mm 5-µm column with a mobile phase contained gradient mixtures, which constitutes of purified water and acetonitrile in the ratio of 700:300 (Port-A) and acetonitrile (Port-B). The optimized flow rate of 2.0 mL/min and UV detection was made at 210 nm and column temperature of 40 °C. The resolution among MP, PP, BHT and ATP was found to be > 4. The optimized method was validated as per the guidelines prescribed by the international conference on harmonization. Regression analysis confers a correlation coefficient higher than 0.999 for MP, PP, BHT and ATP peaks. The % RSD for the interday and intraday precisions for MP, PP, BHT and ATP was estimated and found to be < 5.0. The proposed HPLC method could be suitable for the estimation of these drugs in different types of pharmaceutical drug products. Schematic diagram of the procedure for the simultaneous determination of MP, PP, BHT and ATP contents in marijuana capsules

The effects of varying Mg2+ ion concentrations on contractions to the cotransmitters ATP and noradrenaline in the rat vas deferens.

The vas deferens responds to a single electrical pulse with a biphasic contraction caused by cotransmitters ATP and noradrenaline. Removing Mg2+ (normally 1.2 mM) from the physiological salt solution (PSS) enhances the contraction. This study aimed to determine the effect of Mg2+ concentration on nerve cotransmitter-mediated contractions. Rat vasa deferentia were sequentially bathed in increasing (0, 1.2, 3 mM) or decreasing (3, 1.2, 0 mM) Mg2+ concentrations. At each concentration a single field pulse was applied, and the biphasic contraction recorded. Contractions to exogenous noradrenaline 10 μM and ATP 100 μM were also determined. The biphasic nerve-mediated contraction was elicited by ATP and noradrenaline as NF449 (10 μM) and prazosin (100 nM) completely prevented the respective peaks. Taking the contractions in normal PSS (Mg2+ 1.2 mM) as 100%, lowering Mg2+ to 0 mM enhanced the ATP peak to 170 ± 7% and raising Mg2+ to 3 mM decreased it to 39 ± 3%; the noradrenaline peak was not affected by lowering Mg2+ to 0 mM (97 ± 3%) but was decreased to 63 ± 4% in high Mg2+ (3 mM). Contractions to exogenous ATP, but not noradrenaline, were increased in Mg2+ 0 mM and both were inhibited with Mg2+ 3 mM. Changing Mg2+ concentration affects the contractions elicited by the cotransmitters ATP and noradrenaline. The greatest effects were to potentiate the contraction to ATP in Mg2+ 0 mM and to inhibit the contraction to both ATP and noradrenaline in high Mg2+. Future publications should clearly justify any decision to vary the magnesium concentration from normal (1.2 mM) values.

A day in the life of mitochondria reveals shifting workloads

Mitochondria provide energy for cellular function. We examine daily changing patterns of mitochondrial function and metabolism in Drosophila in vivo in terms of their complex (I-IV) activity, ATP production, glycolysis, and whole fly respiration in the morning, afternoon and night. Complex activity and respiration showed significant and unexpected variation, peaking in the afternoon. However, ATP levels by contrast are >40% greater in the morning and lowest at night when glycolysis peaks. Complex activity modulation was at the protein level with no evidence for differential transcription over the day. Timing differences between increased ATP production and peaks of complex activity may result from more efficient ATP production early in the day leaving complex activity with spare capacity. Optical stimulation of mitochondria is only possible in the mornings when there is such spare capacity. These results provide first evidence of shifts in cellular energy capacity at the organism level. Understanding their translation may be significant to the chosen timing of energy demanding interventions to improve function and health.

Donor Small-Droplet Macrovesicular Steatosis Affects Liver Transplant Outcome in HCV-Negative Recipients.

Background No data are available on liver transplantation (LT) outcome and donor liver steatosis, classified as large droplet macrovesicular (Ld-MaS), small-droplet macrovesicular (Sd-MaS), and true microvesicular (MiS), taking into account the recipient Hepatitis C virus (HCV) status. Aim We investigate the impact of allograft steatosis reclassified according to the Brunt classification on early graft function and survival after LT. Methods We retrospectively reviewed 204 consecutive preischemia biopsies of grafts transplanted in our center during the period 2001-2011 according to recipient HCV status. Results The median follow-up after LT was 7.5 years (range: 0.0-16.7). In negative recipients (n=122), graft loss was independently associated with graft Sd-MaS, in multivariable Cox regression models comprehending only pre-/intraoperative variables (HR=1.03, 95%CI=1.01-1.05; P=0.003) and when including indexes of early postoperative graft function (HR=1.04, 95%CI=1.02-1.06; P=0.001). Graft Sd-MaS>15% showed a risk for graft loss > 2.5-folds in both the models. Graft Sd-MaS>15% was associated with reduced graft ATP content and, only in HCV- recipients, with higher early post-LT serum AST peaks. Conclusions In HCV-negative recipients, allografts with >15% Sd-MaS have significantly reduced graft survival and show low ATP and higher AST peaks in the immediate posttransplant period. Donors with >15% Sd-MaS have significantly higher BMI, longer ICU stays, and lower PaO2.

Alteration in nerves and neurotransmitter stimulation of lacrimal gland secretion in the TSP-1-/- mouse model of aqueous deficiency dry eye.

The purpose of this study is to determine neural, vascular, protein secretion, and cellular signaling changes with disease progression in lacrimal glands of the thrombospondin-1−/− (TSP-1−/−) mouse model of dry eye compared to C57BL/6 wild type (WT) mice. Neural innervation was reduced in TSP-1−/− lacrimal glands compared to WT controls, whereas the number of blood vessels was increased. Intracellular Ca2+ stores and the amount of lysosomes, mitochondria and secretory granules, but not the endoplasmic reticulum, were reduced in TSP-1−/− compared to WT acini at 12 weeks of age. Ex-vivo high KCl-evoked secretion was decreased in TSP-1−/− compared to WT lacrimal gland tissue pieces. The α1D-adrenergic agonist-stimulated response was increased in TSP-1−/− at 4 and 24 weeks but decreased at 12 weeks, and the ATP and MeSATP-stimulated peak [Ca2+]i responses were decreased at 24 weeks. These changes were observed prior to appearance of mononuclear infiltrates. We conclude that in the lacrimal gland the absence of TSP-1: injures peripheral nerves; blocks efferent nerve activation, decreases protein secretion, and, alters intracellular Ca2+ stores. Through these effects the absence of TSP-1 leads to disruption of ocular surface homeostasis and development of dry eye.

Preliminary Study on Hepatocyte-Targeted Phosphorus-31 MRS Using ATP-Loaded Galactosylated Chitosan Oligosaccharide Nanoparticles

Background. The clinical applications of hepatic phosphorus-31 magnetic resonance spectroscopy (31P MRS) remain to be difficult because the changes of phosphates between normal hepatic tissues and pathological tissues are not so obvious, and furthermore, up to now there is few literature on hepatocyte-targeted 31P MRS. Materials and Methods. The ATP-loaded Gal-CSO (Gal-CSO/ATP) nanoparticles were prepared and the special cellular uptake of them as evaluated by using HepG-2 tumor cells and A549 tumor cells, respectively. Two kinds of cells were incubated with the nanoparticles suspension, respectively. Then were prepared the cell samples and the enhancement efficiency of ATP peaks detected by 31P MRS was evaluated. Results. The cellular uptake rate of Gal-CSO/ATP nanoparticles in HepG-2 cells was higher than that in A549 cells. Furthermore, the enlarged ATP peaks of Gal-CSO/ATP nanoparticles in HepG-2 cells were higher than those in A549 cells in vitro detected by 31P MRS. Conclusions. Gal-CSO/ATP nanoparticles have significant targeting efficiency in hepatic cells in vitro and enhancement efficiency of ATP peaks in HepG-2 cells. Furthermore, 31P MRS could be applied in the research of hepatic molecular imaging.

Exercise efficiency is reduced by mitochondrial uncoupling in the elderly

A reduction in exercise efficiency accompanies ageing in humans. Here we evaluated the impact of changes in the contractile-coupling and mitochondrial-coupling efficiencies on the reduction in exercise efficiency in the elderly. Nine adult (mean, 38.8 years old) and 40 elderly subjects (mean, 68.8 years old) performed a cycle ergometer test to measure O2 uptake and leg power output up to the aerobic limit ( ). Reduced leg power output per unit O2 uptake was reflected in a drop in delta efficiency (εD) from 0.27 ± 0.01 (mean ± SEM) in adults to 0.22 ± 0.01 in the elderly group. Similar declines with age were apparent for both the leg power output at and the ATP generation capacity (ATPmax) determined in vivo using (31)P magnetic resonance spectroscopy. These similar declines resulted in unchanged contractile-coupling efficiency values (εC) in the adult (0.50 ± 0.05) versus the elderly group (0.58 ± 0.04) and agreed with independent measures of muscle contractile-coupling efficiency in human quadriceps (0.5). The mitochondrial-coupling efficiency calculated from the ratio of delta to contractile-coupling efficiencies in the adults (εD/εC = 0.58 ± 0.08) corresponded to values for well-coupled mitochondria (0.6); however, εD/εC was significantly lower in the elderly subjects (0.44 ± 0.03). Conversion of ATPmax per mitochondrial volume (ATPmax/Vv[mt,f]) reported in these groups into thermodynamic units confirmed this drop in mitochondrial-coupling efficiency from 0.57 ± 0.08 in adults to 0.41 ± 0.03 in elderly subjects. Thus, two independent methods revealed that reduced mitochondrial-coupling efficiency was a key part of the drop in exercise efficiency in these elderly subjects and may be an important part of the loss of exercise performance with age.

The reproducibility of measurements of intramuscular magnesium concentrations and muscle oxidative capacity using 31P MRS.

31P magnetic resonance spectroscopy (31P MRS) has been used to measure intramuscular magnesium concentrations and muscle metabolism. Abnormal intramuscular magnesium has been reported in several patient populations with suspected metabolic disorders. The purpose of this study was to evaluate our ability to measure intramuscular magnesium and muscle metabolism in the quadriceps muscles of healthy subjects, and to test whether these measurements were influenced by prior exercise. Twelve normal, healthy male volunteers were tested in a 3 Tesla magnet on four separate days. Resting [Mg2+] was calculated from the heights and frequency shifts of the phosphate, phosphocreatine and ATP peaks. Phosphocreatine (PCr) recovery kinetics were measured after 30-39 second bouts of isometric exercise. Thirty minutes prior to the 3rd test session the subjects completed a 2 hour treadmill walk at 40-60% of heart rate reserve. Resting [Mg2+] averaged 0.388 mM and had an interclass correlation coefficient between days (ICC) of 0.352. The mean end exercise PCr was 47.6% and the mean end exercise pH was 6.97. PCr recovery averaged 39 seconds (p = 0.892) and had an ICC of 0.819. Prior long duration exercise did not produce significant alterations in either PCr recovery kinetics or intracellular magnesium levels (p = 0.440). In conclusion, the reproducibility of Resting [Mg2+] was less than that of PCr recovery measurements, and may reflect the sensitivity of these measurements to phasing errors. In addition, prior exercise is unlikely to alter measurements of resting metabolites or muscle metabolism suggesting that rigorous control of physical activity prior to metabolic testing is unnecessary.

Bioluminescent assay of microbial ATP in postmortem tissues for the estimation of postmortem interval

To study the relationship between changes of microbial ATP in four kinds of murine tissues and the postmortem interval (PMI), healthy SD rats were sacrificed and their muscles, livers, spleens and kidneys were sampled at different postmortem intervals. The concentration of microbial ATP was detected using bioluminescent assay and the data was statistically analyzed. The concentration of microbial ATP in muscle increased with PMI time. The peak appeared at the 7th day after death, and at the 10th day, microbial ATP in muscle tissue increased again. In internal organs, the peaks of microbial ATP were observed at the 8th day after death and the level decreased during 8-10 d. The differences in microbial ATP concentration in liver, spleen and kidney were not statistically significant. During day 0 to day 9 after death, the correlation was best between PMI and microbial ATP in muscle. With PMI as the independent variable, the cubic polynomial regression equation was Y=0.02X(3)-0.166X(2)-0.666X+13.412 (R (2)=0.989, P<0.01). In internal organs, the best correlation was found between PMI and microbial ATP during day 0 to day 10. With PMI as the independent variable, the cubic polynomial regression equation was Y=0.016X(3)-0.127X(2)-0.809X+13.324 (R (2)=0.986, P<0.01). There existed high correlations between PMI and microbial ATP concentration in rat tissues. Since only a small amount of tissue was needed for the detection and the sample was not affected by self-decomposition, the method may extend the time range of PMI estimation.

ATP release by cardiac myocytes in a simulated ischaemia model: Inhibition by a connexin mimetic and enhancement by an antiarrhythmic peptide

We studied the role of connexin hemichannels in the release of ATP by neonatal cardiac myocytes subject to ischaemic stress. Mechanical, osmotic and oxidative stress and changes in extracellular or intracellular Ca 2+ levels induce connexin hemichannels located in the plasma membrane to open and release small ions and molecules with signaling potential such as ATP. Since ATP release has been implicated in adaptation to oxygen deprivation, we studied its release by cardiac myocytes incubated in a custom-built hypoxia chamber for various periods. In a simulated ischaemia model (0.5% oxygen and 0.2 g/l glucose) a peak of ATP release occurred at 80 min followed by a return to steady state levels for a further 200 min. This peak of ATP release was not observed in myocytes subject to hypoxia (0.5% oxygen, 3.0 g/l glucose). ATP release in ischaemia was influenced by two classes of reagents that target connexins, the channel forming proteins of gap junctions. First, the connexin hemichannel inhibitors Gap 26 and 18a glycyrrhetinic acid abolished the ATP peak of release. Second, the AAP10, a peptide with antiarrhythmic properties markedly increased the peak of ATP release observed at 80 min of ischaemia and also induced a second smaller peak at 180–240 min. ATP content of the myocytes and Cx43 phosphorylation were monitored. Since the release of ATP in ischaemia was abolished by connexin channel inhibitors and stimulated by a peptide developed to target connexins in the context of cardiac arrhythmia, the results suggest that nucleotide release by connexin hemichannels is likely to feature in the response of myocytes to ischaemic stress in the heart.

Simultaneous Determination of Adenosine and Adenosine‐5′‐triphosphate at Nanogold Modified Indium Tin Oxide Electrode by Osteryoung Square‐Wave Voltammetry

AbstractSimultaneous determination of adenosine and adenosine‐5′‐triphosphate has been described using nanogold modified indium tin oxide electrode. Gold nanoparticles catalyze adenosine oxidation which results in increasing separation of oxidation peaks of adenosine and ATP, making it possible to determine adenosine and adenosine‐5′‐triphosphate simultaneously. The detection limits for adenosine and ATP were found as 0.07 μM and 0.10 μM respectively with sensitivity 22.9 nA μM−1 and 20.9 nA μM−1. The proposed method was also used for sensing the compounds in biological samples. Influence of various square‐wave parameters and different pH conditions on peak current has also been reported.

Increase of free Mg2+in the skeletal muscle of chronic fatigue syndrome patients

In a previous study we evaluated muscle blood flow and muscle metabolism in patients diagnosed with chronic fatigue syndrome (CFS). To better understand muscle metabolism in CFS, we re-evaluated our data to calculate free Magnesium levels in skeletal muscle. Magnesium is an essential cofactor in a number of cell processes. A total of 20 CFS patients and 11 controls were evaluated. Phosphorus magnetic resonance spectroscopy from the medial gastrocnemius muscle was used to calculate free Mg2+ from the concentrations and chemical shifts of Pi, PCr, and beta ATP peaks. CFS patients had higher magnesium levels in their muscles relative to controls (0.47 + 0.07 vs 0.36 + 0.06 mM, P < 0.01), although there was no difference in the rate of phosphocreatine recovery in these subjects, as reported earlier. This finding was not associated with abnormal oxidative metabolism as measured by the rate of recovery of phosphocreatine after exercise. In summary, calculation of free Mg2+ levels from previous data showed CFS patients had higher resting free Mg2+ levels compared to sedentary controls.

Analytical Biochemcial Analysis of Commerical Defibrotide - Sequence Analysis of the Nucleotidic Fractions Via Delayed Extraction Matrix Assissted Laser Desorption / Ionization Time of Flight Mass Spectrometry.

Introduction: Defibrotide (DF), now in clinical trials in Veno-Occlussive Disease (VOD), and Multi-Organ Failure (MOF) as an anti-thrombotic agent is also recognized as a modulator of phosphorilated nucleotides of ATP, ADP, NAD/NADH and cAMP..Thus far the mechanism of its polypharmacology, and the.identity of its biologically active components are not known.The precursor molecule is a double stranded mammalian DNA. And the final commercial product is a mixture of nucleotidic fractions which have formed by the ability of the molecule to form intra-molecular bonding., with random sequences such as:P1-5, (dAp)12–24, (dGp) 10–20, (dTp)13–26, (dCp)10–20, Wherein, P=phosphoric radical; dAp= deoxyadenylic monomer; dGp= deoxyguanylic monomer; dTp= deoxythymidylic monomer; dCp= deoxycytidilic monomer.(US Patent # 4,985,552,Crinos). This report aims to investigate for the first time the sequence spesificity, and the reproducability of the nucleotidic fractions of DF. and to define a structural base for DF as a global modulator of the signalling network.Method:.Materials from two commercial forms of DF, Prociclide and Noravid in solution and capsule forms, were separated in each case by HPLC on a Vydac, C8 column using 0.1% trifluoroacetate in water. 513 mass spectra peaks were collected, freeze dried, preserved on ice and subjected to Voyager Elite Biospectrometry Workstation using the technique of delayed extraction matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry..A blank run representing DNA matrix was done in each case on MALDI-TOF to detect the low MW particles that were not related to the sample. The peaks were superimposed on the HPLC profiles of mono-, di-, tri-phosphates of T,C,G, A via crossmatching the MW, together with retention times as described above.260,000 crossmatches were done.Statistics: The min, max, mean and median mass values were stratified into four respective batches of DF, capsule and liquid forms, and two commercial labels The standard deviations (SD) were calculated in each of the above stratifications with the “n-1” method.Results: HPLC profiles contained more than one molecular species with MWs ranging from <300 to <1500 Da. and were not superimposable. A representative panel would include deoxyadenylic and deoxycytidylic monomers, guanine and other nucleotides of 3–25 bases, such as dC,dA, G, dGMP, AMP, oligonucleotides with 3–5 bases such as UTP, dTTP, CTP, ATP, dGTP, cTMP, CMP, cGMP, and dAMP. Since the study was only directed at bases, deoxy-bases and mono-and di-, and tri-nucleotides of </= 560 Da (the calculated MW of alkali sodium salt of ATP), statistical calculations were carried out only for these fractions. When compared to capsules, liquid DF showed double number of peaks of ATP, ADP, AMP, GTP, GDP, and GMP. The standard deviations (STD) on the nucleotides in the various liquid forms of Defibrotide were much narrower, ranging between (+ / −) 0–1.4, capsule forms showing less consistency with STD's of (+ / −)0–2.1Conclusion: The sequence analysis of the nucleotidic fractions of <560 D MW displayed presence of cAMP, ATP, ADP, GTP, GDP,GMP, CTP,CMP,CDP, TTP,TDP,TMP reproducably between batches. DF maybe a direct and indirect supplier of messenger nucleotides. This structural analysis may support this hypothesis generating report presenting DF as the prototype global modulator of the signalling network as a mechanism of its polypharmacology.

Skeletal muscle metabolic and ionic adaptations during intense exercise following sprint training in humans.

The effects of sprint training on muscle metabolism and ion regulation during intense exercise remain controversial. We employed a rigorous methodological approach, contrasting these responses during exercise to exhaustion and during identical work before and after training. Seven untrained men undertook 7 wk of sprint training. Subjects cycled to exhaustion at 130% pretraining peak oxygen uptake before (PreExh) and after training (PostExh), as well as performing another posttraining test identical to PreExh (PostMatch). Biopsies were taken at rest and immediately postexercise. After training in PostMatch, muscle and plasma lactate (Lac(-)) and H(+) concentrations, anaerobic ATP production rate, glycogen and ATP degradation, IMP accumulation, and peak plasma K(+) and norepinephrine concentrations were reduced (P<0.05). In PostExh, time to exhaustion was 21% greater than PreExh (P<0.001); however, muscle Lac(-) accumulation was unchanged; muscle H(+) concentration, ATP degradation, IMP accumulation, and anaerobic ATP production rate were reduced; and plasma Lac(-), norepinephrine, and H(+) concentrations were higher (P<0.05). Sprint training resulted in reduced anaerobic ATP generation during intense exercise, suggesting that aerobic metabolism was enhanced, which may allow increased time to fatigue.

Ca2+ and cAMP-activated Cl- conductances mediate Cl- secretion in a mouse renal inner medullary collecting duct cell line.

1. The nature of Cl- conductance(s) participating in transepithelial anion secretion by renal inner medullary collecting duct (IMCD, mIMCD-K2 cell line) was investigated. 2. Extracellular ATP (100 microM) stimulated a transient increase in both whole-cell Cl- conductance and intracellular free Ca2+. In contrast, ionomycin (10-100 nM) caused a sustained increase in whole-cell Cl- conductance. Pre-loading cells with the Ca2+ buffer BAPTA abolished the ATP-dependent responses and delayed the onset of the increase observed with ionomycin. 3. The Ca2+-activated whole-cell Cl- current stimulated by ATP (peak) and ionomycin (maximal) displayed (i) a linear steady-state current-voltage relationship and (ii) time and voltage dependence with slow activation at +80 mV and slow inactivation at -80 mV. In BAPTA-loaded cells, ionomycin-elicited whole-cell currents exhibited pronounced outward rectification with time-dependent activation/inactivation. 4. Ca2+-activated and forskolin-activated Cl- conductances co-exist since ATP activation of whole-cell current occurred during a maximal stimulation by forskolin in single cell recordings. 5. In IMCD epithelial layers, ATP and ionomycin stimulated an inward short circuit current (Isc) dependent upon basal medium Na+ and Cl-/HCO3- but independent of the presence of apical bathing medium Na+ and Cl-/HCO3-. This was identical to forskolin stimulation and consistent with transepithelial anion secretion. 6. PCR amplification of reverse-transcribed mRNA using gene-specific primers demonstrated expression of both cystic fibrosis transmembrane conductance regulator (CFTR) mRNA and Ca2+-activated Cl- channel (mCLCA1) mRNA in mIMCD-K2 cells. 7. Ca2+ and forskolin-activated Cl- conductances participate in anion secretion by IMCD.

Mg‐Atp Binding: Its Modification by Spermine, the Relevance to Cytosolic Mg2+ Buffering, Changes in the Intracellular Ionized Mg2+ Concentration and the Estimation of Mg2+ by 31P‐NMR

It is now generally accepted that the intracellular ionized magnesium concentration ([Mg2+]i) in muscle cells is around 1 mmol l−1; in heart muscle this means that from the total some 90‐95% is bound (see McGuigan et al. 1991a). Although binding will include sequestration by intracellular organelles, a large part of the binding is by ATP in the cytosol and an equilibrium exists in the cytosol between free ATP, ionized magnesium and Mg‐ATP. The extend of this equilibrium depends on the equilibrium constant of the reaction, which is a function of pH, temperature and ionic strength. This equilibrium constant is also important in the estimation of [Mg2+]i using 31P‐NMR. In this method the difference between the α and β peaks of ATP is measured and from this shift and the equilibrium constant between Mg2+ and ATP in the cytosol, the [Mg2+]i can be calculated (Vink, 1993).

Mg-ATP binding: its modification by spermine, the relevance to cytosolic Mg2+ buffering, changes in the intracellular ionized Mg2+ concentration and the estimation of Mg2+ by 31P-NMR

It is now generally accepted that the intracellular ionized magnesium concentration ([Mg2+]i) in muscle cells is around 1 mmol l-1; in heart muscle this means that from the total some 90-95 % is bound (see McGuigan et al. 1991a). Although binding will include sequestration by intracellular organelles, a large part of the binding is by ATP in the cytosol and an equilibrium exists in the cytosol between free ATP, ionized magnesium and Mg-ATP. The extend of this equilibrium depends on the equilibrium constant of the reaction, which is a function of pH, temperature and ionic strength. This equilibrium constant is also important in the estimation of [Mg2+]i using 31P-NMR. In this method the difference between the α and β peaks of ATP is measured and from this shift and the equilibrium constant between Mg2+ and ATP in the cytosol, the [Mg2+]i can be calculated (Vink, 1993).

The effect of the neuropeptide substance P on desensitization of ATP receptors of PC12 cells.

1. Patch clamp recording (whole cell configuration) was employed to investigate the modulatory action of substance P on inward currents elicited by adenosine 5'-triphosphate (ATP, focally applied via a pressure pipette) from phaechromocytoma (PC12) cells usually held at -70 mV. 2. Bath-applied substance P (0.2-20 microM) had no effect on baseline membrane current but reversibly reduced ATP peak currents in a concentration-dependent fashion. The depressant effect was not associated with a change in the ATP current reversal potential. 3. Equiamplitude peak responses induced by 50 microM or 5 mM ATP were differentially affected by substance P which preferentially reduced currents evoked by 5 mM ATP. In the presence of substance P a conditioning pulse of ATP evoked a stronger depression to subsequent test pulses of the same agonist. 4. Combined patch clamping and confocal laser imaging of intracellular Ca2+ ([Ca2+]i) of single PC12 cells showed that substance P (applied by a pressure pipette) per se had no effect on [Ca2+]i or current baseline, although it reduced the inward current and associated [Ca2+]i rise elicited by ATP. 5. These results are interpreted as due to facilitation by substance P of desensitization of ATP-gated P2x2 receptors of PC12 cells. It is proposed that the novel modulation by this peptide of ATP responses may serve as a model for further studies aimed at elucidating the action of substance P on purinergic neurotransmission.

Post-ischemic 31P NMR determination of myocardial intracellular pH in vivo using ATP peak.

31P NMR allows non-invasive measurement of intracellular pH, which drops during tissue hypoxia or ischemia. Determination is usually based on the chemical shift between the inorganic phosphate (P(i)) and phosphocreatine (PCr) peaks. During reperfusion, P(i) is taken up to form PCr and ATP, and in our model at least (an isolated, working rat heart perfused with an erythrocyte suspension), the level of P(i) reduces well below the pre-ischemic level, making pH determination difficult. The chemical shifts of the three ATP peaks also depend on pH, and the level of ATP remains high during reperfusion, so these might be used to determine pH. The results of one experiment are presented in detail, showing the time course of high energy phosphate levels before, during and after a 32 min ischemic insult, and close agreement between the pH determinations from the Pi and gamma-ATP peaks can be seen. The formula used to calculate pH from the ATP peak was: pH (ATP) = 0.59 delta 2-5.0 delta + 15.9 where delta is the shift in ppm between PCr and gamma-ATP. All pH readings by both methods from a series of seven experiments were compared and a 1:1 agreement demonstrated (correlation coefficient 0.63, p < 0.0001). Although the ATP shifts also depend on magnesium complexation which we have ignored, this appears to be justifiable within the errors of the method; the good agreement between the results of the two methods, and the ability to determine pH during reperfusion suggest that calculation of intracellular pH from the chemical shift of gamma-ATP is a useful technique.