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PubMed2005

Erythrocyte magnesium fluxes in mice with nutritionally and genetically low magnesium status.

Low intracellular magnesium (Mg) contents may be observed in case of severe Mg insufficient intake or because of genetic regulation. This work was conducted to investigate the influence of intracellular Mg content on erythrocyte Mg(2+) influx and efflux in mice with low nutritionally and genetically (MGL and MGH mice) Mg status. C57BL6 mice were fed for 2 wks a diet containing 1000 mg Mg/kg diet Mg (control group), 100 mg Mg/kg diet (Mg-marginal group) or 30 mg Mg/kg diet (Mg deficient group), while mice with low (MGL) and high (MGH) Mg levels were fed a control diet for 2 wks. The quantification of erythrocyte Mg(2+) influx and efflux was performed using a stable isotope of Mg. Our results showed that erythrocyte Mg(2+) influx and efflux were respectively increased and decreased in nutritional Mg deficiency; while in genetically determined Mg status Mg(2+) fluxes were lower in MGL mice compared to MGH mice. Moreover Mg(2+) efflux was significantly correlated to Mg level in erythrocytes in all the mice studied (p < 0.001). In conclusion, erythrocyte Mg(2+) influx and efflux are modulated by low Mg status, namely decreased Mg(2+) efflux compensate for nutritional Mg deficiency, while the genetic regulation of erythrocyte Mg(2+) content depends on modification of Mg(2+) influx.

Animals↗

PubMed2025

Somatic Mutations in MCOLN3 Are Associated With Aldosterone-Producing Adenomas.

BACKGROUND: Primary aldosteronism is a common but underdiagnosed cause of endocrine hypertension that contributes to global cardiovascular morbidity and mortality. It is characterized by renin-independent hyperaldosteronism that originates from adrenal lesions-the majority of which are found to harbor aldosterone-driver somatic mutations in genes encoding ion-transporting proteins. These mutations disrupt intracellular calcium homeostasis, facilitating a pathological increase in aldosterone synthase expression and aldosterone production. Elucidating the exact mechanisms causing aldosterone excess in primary aldosteronism would further the development of targeted treatments and alleviate the global hypertension burden. METHODS: Next-generation sequencing analysis of formalin-fixed paraffin-embedded aldosterone-producing adenomas identified novel somatic variants in MCOLN3 (encoding the cation-permeable channel, TRPML3). Electrophysiological, fura-2 calcium measurements, gene expression, and steroid quantification studies were performed in adrenal HAC15 cells to characterize the functional effects of the novel MCOLN3 mutations. RESULTS: Three somatic MCOLN3 variants (p.Y391D, p.F415I, and p.N411_V412delinsI) were identified in aldosterone-producing adenomas from 4 male primary aldosteronism patients. Mutated MCOLN3 expressed in HAC15 cells resulted in a gain-of-function phenotype, which induced cell membrane depolarization and calcium influx and, in turn, triggered a significant increase in aldosterone synthase expression and aldosterone production. CONCLUSIONS: This is the first report of disease-causing MCOLN3 mutations in humans and the first to implicate mutated MCOLN3 as a driver of dysregulated aldosterone production in primary aldosteronism.

Humans↗

PubMed2001

A functional enhanced green fluorescent protein (EGFP)-tagged angiotensin II at(1a) receptor recruits the endogenous Galphaq/11 protein to the membrane and induces its specific internalization independently of receptor-g protein coupling in HEK-293 cells.

The angiotensin II (Ang II) AT(1A) receptor was tagged at its C terminus with the enhanced green fluorescent protein (EGFP), and the corresponding chimeric cDNA was expressed in HEK-293 cells. This tagged receptor presents wild-type pharmacological and signaling properties and can be immunodetected by Western blotting and immunoprecipitation using EGFP antibodies. Therefore, this EGFP-tagged AT(1A) receptor is the perfect tool for analyzing in parallel the subcellular distributions of the receptor and its interacting G protein and their trafficking using confocal microscopy. Morphological observation of both the fluorescent receptor and its cognate Galphaq/11 protein, identified by indirect immunofluorescence, and the development of a specific software for digital image analysis together allow examination and quantification of the cellular distribution of these proteins before and after the binding of different agonist or antagonist ligands. These observations result in several conclusions: 1) Expression of increasing amounts of the AT(1A) receptor at the cell surface is associated with a progressive recruitment of the cytosolic Galphaq/11 protein at the membrane; 2) Internalization of the EGFP-tagged AT(1A) induced by peptide ligands but not nonpeptide ligands is accompanied by a Galphaq/11 protein intracellular translocation, which presents a similar kinetic pattern but occurs predominantly in a different compartment; and 3) This Galphaq/11 protein cellular translocation is dependent on receptor internalization process, but not G protein coupling and signal transduction mechanisms, as assessed by pharmacological data using agonists and antagonists and the characterization of AT(1A) receptor mutants (D(74)N and Delta329) for which the coupling and internalization functions are modified.

Angiotensin II↗

PubMed2000

Chemical and physiological characterization of fluo-4 Ca(2+)-indicator dyes.

We have developed fluo-4, a new fluorescent dye for quantifying cellular Ca2+ concentrations in the 100 nM to 1 microM range. Fluo-4 is similar in structure and spectral properties to the widely used fluorescent Ca(2+)-indicator dye, fluo-3, but it has certain advantages over fluo-3. Due to its greater absorption near 488 nm, fluo-4 offers substantially brighter fluorescence emission when used with excitation by argon-ion laser or other sources in conjunction with the standard fluorescein filter set. In vitro, fluo-4 exhibited high fluorescence emission, a high rate of cell permeation, and a large dynamic range for reporting [Ca2+] around a Kd(Ca2+) of 345 nM. We have also developed several Ca(2+)-indicators related to fluo-4 having lower affinities for Ca2+ that are useful in cellular studies requiring quantification of higher [Ca2+]. In a variety of physiological studies of live cells, fluo-4 labeled cells more brightly than did fluo-3, when challenged with procedures designed to elevate calcium levels. Fluo-4 is well suited for photometric and imaging applications that make use of confocal laser scanning microscopy, flow cytometry, or spectrofluorometry, or in fluorometric high-throughput microplate screening assays. Because of its higher fluorescence emission intensity, fluo-4 can be used at lower intracellular concentrations, making its use a less invasive practice.

3T3 Cells↗

PubMed1995

Adjustment of K' to varying pH and pMg for the creatine kinase, adenylate kinase and ATP hydrolysis equilibria permitting quantitative bioenergetic assessment.

Physiologists and biochemists frequently ignore the importance of adjusting equilibrium constants to the ionic conditions of the cell prior to calculating a number of bioenergetic and kinetic parameters. The present study examines the effect of pH and free magnesium levels (free [Mg2+]) on the apparent equilibrium constants (K') of creatine kinase (ATP: creatine N-phosphotransferase; EC 2.7.3.2), adenylate kinase (ATP:AMP phosphotransferase; EC 2.7.4.3) and adenosinetriphosphatase (ATP phosphohydrolase; EC 3.6.1.3) reactions. We show how K' can be calculated using the equilibrium constant of a specified chemical reaction (Kref) and the appropriate acid-dissociation and Mg(2+)-binding constants at an ionic strength (I) of 0.25 mol l-1 and 38 degrees C. Substituting the experimentally determined intracellular pH and free [Mg2+] into the equation containing a known Kref and two variables, pH and free [Mg2+], enables K' to be calculated at the experimental ionic conditions. Knowledge of K' permits calculation of cytosolic phosphorylation ratio ([ATP]/[ADP][Pi]), cytosolic free [ADP], free [AMP], standard transformed Gibbs energy of formation (delta fG' degrees ATP) and the transformed Gibbs energy of the system (delta fG' ATP) for the biological system. Such information is vital for the quantification of organ and tissue bioenergetics under physiological and pathophysiological conditions.

Adenosine Triphosphate↗

PubMed2003

Subicular dendritic arborization in Alzheimer's disease correlates with neurofibrillary tangle density.

Intracellular accumulation of PHFtau in Alzheimer's disease (AD) disrupts the neuronal cytoskeleton and other neuronal machinery and contributes to axonal and dendritic degeneration, and neuronal death. Furthermore, amyloid-beta (Abeta) has been reported to be toxic to neurons and neurites. While loss of presynaptic elements is an established feature of AD, the nature and extent of dendritic degeneration has been infrequently studied. We investigated MAP2-immunoreactive dendrites using a novel method of high-throughput quantification and also measured cortical thickness and the densities of NeuN-immunoreactive neurons, PHFtau neurofibrillary tangles (NFTs), and Abeta plaque burden in the subiculum in AD and elderly controls. Corrected for atrophy, the "dendritic arborization index" was significantly reduced by up to 66% in all three layers of the subiculum. Laminar thickness was reduced by an average 33% and there was a marked reduction in neuron density of approximately 50%. As expected, NFTs and Abeta plaques were significantly increased in AD. Dendritic arborization indices negatively correlated with NFT densities while no significant correlations were found with Abeta plaque densities. The pattern of dendritic loss in the subiculum and the correlations with NFT densities respectively suggest that deafferentation and intrinsic neurofibrillary degeneration both may contribute to dendritic loss in AD.

Aged↗

PubMed1996

Ca2+ waves in lung capillary endothelium.

Although cytosolic Ca2+ importantly regulates organ function, lung microvascular [Ca2+]i regulation remains poorly understood because of the lack of direct in situ quantification. In the present study, we report the first endothelial [Ca2+]i quantification by the fura 2 method in microscopically imaged venular capillaries of the isolated blood-perfused rat lung. Sequential images indicated the presence of intercellular Ca2+ waves that spontaneously originated from pacemaker endothelial cells and then spread for short distances along the capillary wall, inducing synchronous endothelial [Ca2+]i oscillations. Fast Fourier analyses of the oscillations revealed a dominant wave component with an amplitude of 37 nmol/L, frequency of 0.4 min-1, and velocity of 5 microns/s. The intracellular Ca2+ wave was unaffected by blood flow stoppage or by infusions of Ca(2+)-containing or Ca(2+)-free dextran. Inhibition of the wave by thapsigargin in Ca(2+)-free dextran and by the gap junction uncoupler, heptanol, indicated that it was generated by endosomal Ca2+ release in the pacemaker cell and was propagated by gap junctional communication. In the presence of histamine, enhancement of the wave accounted for a significant component of the coordinated [Ca2+]i increase in the capillary segment. No intercellular Ca2+ waves were evident in adjoining alveolar epithelial cells. Our findings indicate a novel mechanism of [Ca2+]i regulation in the lung capillary under both resting and stimulated conditions. Pacemaker-induced Ca2+ waves, generated intracellularly by unknown initiating mechanisms, communicated to adjoining cells to determine [Ca2+]i profiles in short interbranch segments of capillary walls.

Animals↗

PubMed2001

5-HT-receptor-induced changes of the intracellular cAMP level monitored by a hyperpolarization-activated cation channel.

The HvCNG channel from the moth Heliothis virescens is highly sensitive to cAMP concentrations ranging between 0.1 microM and 5 microM. This HvCNG channel was over-expressed in Spodoptera frugiperda (Sf.9) cells to measure endogenous cAMP levels. Hyperpolarization-activated inward currents were measured in the whole-cell patch-clamp configuration with pipettes filled with different cAMP concentrations to calibrate the system. Varying the cAMP concentration between 0 microM and 100 microM in the pipette, the half-maximal activation voltage ( V1/2) was shifted by +28.5+/-1.7 mV. The activation time constant (tau(a)) was used as a parameter for cAMP quantification because it was independent of the expression level of HvCNG channels. tau(a) changed from 1106+/-60 ms at 0 microM cAMP to 265+/-7 ms at a saturating concentration of 1 mM cAMP. A dose-response relationship yielded values of 0.6 microM for the half-maximal cAMP concentration and 1.5 for the Hill coefficient. Activation of endogenous adenylyl cyclases by 50 microM forskolin induced an elevation of the cAMP level by about 1.6+/-0.2 microM. Co-expressions of HvCNG channels in combination with the mouse 5-HT4a- or 5-HT1A- receptors and the corresponding Gs- or Gi-proteins were successful and allowed us to also verify receptor-induced changes of the cAMP level. Stimulation of m5-HT4a-receptors by 0.1 microM 5-HT induced an increase of cAMP of about 4.6+/-1.5 microM, whereas cAMP levels decreased from a control value of 1+/-0.2 microM to 0.41+/-0.1 microM after stimulation of the m5-HT1A-receptors.

Adenylyl Cyclases↗

PubMed1987

Quantitative study of tissue collagen metabolism.

A procedure for the quantification of various parameters of metabolism of collagen in fibrotic mouse liver has been developed. The method involves derivatization of hydroxyproline, a marker of collagen, with dansyl chloride, high-performance liquid chromatography of the derivative on an octadecyl C-18 column, and its detection by fluorescence. This assay improves upon existing procedures in several respects: It extends the analysis so that not only the collagen content of the tissue but also the metabolism of collagen is determined at levels found intracellularly. It is sensitive enough to quantify 0.1-10 nmol of hydroxyproline, and it includes three major amino acids (hydroxyproline, glycine, and proline) of collagen and two assay controls; it generates information on both the purity and quantity of collagen in each assay. The determination of specific activity of intracellular free [14C]proline, which is the precursor of protein-bound hydroxyproline, defines the specific activity of [14C]hydroxyproline of collagen converted from precursor residues of [14C]proline by the action of prolyl hydroxylase. The specific activity of [14C]hydroxyproline can be used for the evaluation of collagen synthesis and secretion and intracellular and extracellular degradation of the newly synthesized and secreted collagen in the tissue. The determination of specific activities of [14C]hydroxyproline and [14C]proline and of the ratio of [14C]hydroxyproline to [14C]proline of newly secreted collagen provides information concerning the extent of hydroxylation of [14C]proline residues of newly synthesized collagen.

Animals↗

PubMed1995

Quantification of different transferrin receptor pools in primary cultures of porcine blood-brain barrier endothelial cells.

Distribution of iron in the brain varies with region, cell type, and age. Furthermore, some neurological diseases are accompanied by an abnormal accumulation of iron in specific areas of the CNS. These findings implicate a mobile intracerebral iron pool; however, transport of iron across the blood-brain barrier and its regulation are largely unknown. In an extensive series of experiments in primary cultures of porcine blood-brain barrier endothelial cells, we separately quantified surface-bound and total cellular transferrin receptor pools. Although 90% of all transferrin receptors were located inside the cell, only 10% of these intracellular receptors actively took part in the endocytic cycle. This large "inactive" intracellular transferrin receptor pool could either function as a storage site for spare receptors or be activated by the cell to increase its capacity for iron transport. Data were corrected for nonspecific binding by a separate biochemical assessment using a 100-fold excess of unlabeled ligand. Data were also analyzed in a nonlinear curve-fit program. This resulted in a less elaborate and less biased estimate of nonspecific binding.

Animals↗

PubMed1994

Localization and quantification of the insulin-like growth factor-1 receptor in mouse articular cartilage by confocal laser scanning microscopy.

Insulin-like growth factor-1 (IGF-1) is an essential anabolic growth factor in the regulation of cartilage metabolism and exerts its effects by binding to the IGF-1 Type 1 receptor on the chondrocyte membrane. We have localized and quantified in situ IGF-1 receptor expression in intact articular cartilage of normal mice. The IGF-1 receptor was detected immunohistochemically with antibodies to the IGF-1 receptor and visualized with conventional light microscopy and confocal laser scanning microscopy (CLSM). CLSM analysis enabled us to distinguish IGF-1 receptor immunoreactivity on the chondrocyte cell membrane from intracellular staining. We have established two approaches to quantify in confocal images low levels of fluorescence intensity of the immunolocalized IGF-1 receptor at the chondrocyte membrane, i.e., mean pixel measurement and area measurement. The majority of IGF-1 receptor fluorescence intensity was localized on chondrocytes in the middle and deeper zones of cartilage, whereas chondrocytes in the surface zone exhibited negligible fluorescence. The variable distribution of IGF-1 receptor in chondrocytes of articular cartilage suggests that effects of IGF-1 on chondrocytes may be distinctly heterogeneous in the different mouse articular cartilage zones.

Animals↗

PubMed2003

A replacement of the active-site aspartic acid residue 293 in mouse cathepsin D affects its intracellular stability, processing and transport in HEK-293 cells.

The substitution of an active-site aspartic acid residue by asparagine in the lysosomal protease cathepsin D (CTSD) results in a loss of enzyme activity and severe cerebrocortical atrophy in a novel form of neuronal ceroid lipofuscinosis in sheep [Tyynelä, Sohar, Sleat, Gin, Donnelly, Baumann, Haltia and Lobel (2000) EMBO J. 19, 2786-2792]. In the present study we have introduced the corresponding mutation by replacing aspartic acid residue 293 with asparagine (D293N) into the mouse CTSD cDNA to analyse its effect on synthesis, transport and stability in transfected HEK-293 cells. The complete inactivation of mutant D293N mouse CTSD was confirmed by a newly developed fluorimetric quantification system. Moreover, in the heterologous overexpression systems used, mutant D293N mouse CTSD was apparently unstable and proteolytically modified during early steps of the secretory pathway, resulting in a loss of mass by about 1 kDa. In the affected sheep, the endogenous mutant enzyme was stable but also showed the shift in its molecular mass. In HEK-293 cells, the transport of the mutant D293N mouse CTSD to the lysosome was delayed and associated with a low secretion rate compared with wild-type CTSD. These data suggest that the mutation may result in a conformational change which affects stability, processing and transport of the enzyme.

Animals↗

PubMed2004

Multiphoton-FLIM quantification of the EGFP-mRFP1 FRET pair for localization of membrane receptor-kinase interactions.

We present an improved monomeric form of the red fluorescent protein, mRFP1, as the acceptor in biological fluorescence resonance energy transfer (FRET) experiments using the enhanced green fluorescent protein as donor. We find particular advantage in using this fluorophore pair for quantitative measurements of FRET using multiphoton fluorescence lifetime imaging microscopy (FLIM). The technique was exploited to demonstrate a novel receptor-kinase interaction between the chemokine receptor (CXCR4) and protein kinase C (PKC) alpha in carcinoma cells for both live- and fixed-cell experiments. The CXCR4-EGFP: PKCalpha-mRFP1 complex was found to be localized precisely to intracellular vesicles and cell protrusions when imaged by multiphoton fluorescence-FLIM. A comparison of the FRET efficiencies obtained using mRFP1-tagged regulatory domain or full-length PKCalpha as the acceptor revealed that PKCalpha, in the closed (inactive) form, is restrained from associating with the cytoplasmic portion of CXCR4. Live-cell FLIM experiments show that the assembly of this receptor:kinase complex is concomitant with the endocytosis process. This is confirmed by experimental evidence suggesting that the recycling of the CXCR4 receptor is increased on stimulation with phorbol ester and blocked on inhibition of PKC by bisindolylmaleimide. The EGFP-mRFP1 couple should be widely applicable, particularly to live-cell quantitative FRET assays.

Breast Neoplasms↗

PubMed1992

Methylations in hemoglobin from monozygotic twins discordant for cigarette smoking: hereditary and tobacco-related factors.

Dose monitoring of exogenous methylators by measurement of N-methylvaline in hemoglobin (Hb) is rendered difficult due to a relatively high, variable background in unexposed persons. A kinetic study indicates intracellular S-adenosylmethionine to be a main source of these background methylations. A comparison of twin pairs indicates that the variation in methylvaline levels is partly hereditary (P less than 0.001). Therefore, in a study of monozygotic twin pairs discordant for tobacco smoking the resolving power of the monitoring was increased and in vivo doses of methylators from the smoke could be more easily monitored through their adducts to Hb. Probably, twin studies offer a useful tool for the identification and quantification of electrophiles of endogenous and exogenous origin.

Adult↗

PubMed2004

Quantification of the rat left ventricle force and Ca2+ -frequency relationships: similarities to dog and human.

OBJECTIVE: To measure and quantify the force-frequency (FFR) and Ca(2+)-frequency (CaFR) relationships in isolated rat left ventricular (LV) muscle at physiological heart rates and compare the obtained FFR to that measured in larger mammalian muscle from dog and human using the same experimental protocol. METHODS: Rat papillary muscle was isolated from the LV of adult male Sprague-Dawley rats, and dog and human muscles were from free-wall LV biopsies, loaded with the Ca(2+) indicator Fura-2, allowed to recover from isolation trauma and then subjected to direct electrical stimulation while measuring force production and intracellular Ca(2+) transients. RESULTS: We obtained a positive FFR between 1 and 4 Hz that is qualitatively similar to that found in isolated LV epicardial muscle strips from dogs and humans with normal LV function. The FFR reflects the cytosolic Ca(2+) transients in amplitude. Isoproterenol yielded an enhancement in force, but flattening of the FFR, whereas cyclopiazonic acid caused depression of FFR amplitude without changing frequency-dependent shape. CONCLUSION: We describe an experimental protocol that consistently yields positive FFRs in rat, dog and human LV muscle at stimulation rates between 1 and 4 Hz, without significant qualitative differences. We attribute previously observed negative FFR in rat muscle to an increase in SERCA activity early after excision and preparation of the muscle strips.

Aged↗

PubMed1987

Determination of Krebs cycle metabolic carbon exchange in vivo and its use to estimate the individual contributions of gluconeogenesis and glycogenolysis to overall glucose output in man.

Current isotopic approaches underestimate gluconeogenesis in vivo because of Krebs cycle carbon exchange and the inability to measure intramitochondrial precursor specific activity. We therefore applied a new isotopic approach that theoretically overcomes these limitations and permits quantification of Krebs cycle carbon exchange and the individual contributions of gluconeogenesis and glycogenolysis to overall glucose output. [6-3H]Glucose was infused to measure overall glucose output; [2-14C]acetate was infused to trace phosphoenolpyruvate gluconeogenesis and to calculate Krebs cycle carbon exchange as proposed by Katz. Plasma [14C]3-OH-butyrate specific activity was used to estimate intramitochondrial acetyl coenzyme A (CoA) specific activity, and finally the ratio between plasma glucose 14C-specific activity and the calculated intracellular phosphoenolpyruvate 14C-specific activity was used to determine the relative contributions of gluconeogenesis and glycogenolysis to overall glucose output. Using this approach, acetyl CoA was found to enter the Krebs cycle at twice (postabsorptive subjects) and three times (2 1/2-d fasted subjects) the rate of pyruvate, respectively. Gluconeogenesis in postabsorptive subjects (3.36 +/- 0.20 mumol/kg per min) accounted for 28 +/- 2% of overall glucose output and increased twofold in subjects fasted for 2 1/2-d (P less than 0.01), accounting for greater than 97% of overall glucose output. Glycogenolysis in postabsorptive subjects averaged 8.96 +/- 0.40 mumol/kg per min and decreased to 0.34 +/- 0.08 mumol/kg per min (P less than 0.01) after a 2 1/2-d fast. Since these results agree well with previously reported values for gluconeogenesis and glycogenolysis based on determinations of splanchnic substrate balance and glycogen content of serial liver biopsies, we conclude that the isotopic approach applied herein provides an accurate, noninvasive measurement of gluconeogenesis and glycogenolysis in vivo.

3-Hydroxybutyric Acid↗

PubMedDate not supplied

Targeting of sebocytes by aminolevulinic acid-dependent photosensitization.

Photodynamic therapy using 5-aminolevulinic acid-induced protoporphyrin IX has been developed as a very useful therapeutic modality. Recently, several authors have reported on the efficacy of this procedure for acne. This approach is based on the fact that 5-aminolevulinic acid-induced protoporphyrin IX has strong selectivity for sebaceous glands. We used the immortalized human sebaceous gland cell line SZ95 to investigate cellular mechanisms of photodynamic therapy using 5-aminolevulinic acid-induced protoporphyrin IX. Quantification of induced protoporphyrin IX production showed dependence on the applied 5-aminolevulinic acid dose. When SZ95 sebocytes were differentiated by arachidonic acid treatment, there was no difference between them and the control cells with respect to both the amount of 5-aminolevulinic acid-induced protoporphyrin IX and the phototoxic effects. We altered protoporphyrin IX formation rates by growing cells scattered as single cells in the culture dishes. Single cells produced significantly lower protoporphyrin IX levels than those grown with intercellular contacts. Intracellular localization of protoporphyrin IX was imaged using confocal laser scanning microscopy. The differentiation-specific lipid droplets were virtually excluded from protoporphyrin IX fluorescence. In addition to weak mitochondrial and strong membrane fluorescence, distinctive spots with strong fluorescence were observed. These did not colocalize with fluorescent probes for mitochondria, lysosomes or the Golgi apparati.

Aminolevulinic Acid↗

PubMed1991

Improved ion-pair high-performance liquid chromatographic method for the quantification of a wide variety of nucleotides and sugar-nucleotides in animal cells.

An improved method including extraction procedures is presented for the analysis of nucleotides in suspension-cultivated animal cells. Quantification was performed by ion-pair high-performance liquid chromatography after perchloric acid extraction. It was found that the amount of perchloric acid taken for extraction influenced the yield and that cell washing procedures caused deterioration of the analysis results for triphosphates. More than thirty nucleotides and sugar-nucleotides were separated within 25 min using a Supelcosil reversed-phase column (3 microns) with tetrabutylammonium hydrogensulphate as pairing agent and methanol-pH gradient elution. Cultivated hybridoma cells showed variations in intracellular nucleotide concentrations as well as relative amounts during different growth phases, which could reflect the physiological state of a cell culture.

Adenosine Triphosphate↗