Biomedical subjects

A Klip

Publications and source records attributed to A Klip.

At least 163 records · Page 9Linked to original sources

PubMed1988

Insulin-induced decrease in 5'-nucleotidase activity in skeletal muscle membranes.

Insulin releases inositol phosphoglycans from myocytes in culture [(1986) Science 233, 967-972], which display insulinomimetic activity. Because 5'-nucleotidase is anchored to the membrane through inositol-containing phospholipid glycans, we investigated whether insulin could release the enzyme from the membrane. Membranes prepared from hindquarter muscles of rats perfused with insulin showed a 23% decrease in 5'-nucleotidase activity. Isolated membranes from muscle exposed to insulin in vitro also showed a small but reproducible decrease (9%) in 5'-nucleotidase activity relative to unexposed controls. Phospholipase C from Staphylococcus aureus released 60% of the membrane-bound 5'-nucleotidase. We propose that insulin may activate an endogenous phospholipase C that cleaves phospholipid-glycan-anchored proteins.

5'-Nucleotidase↗

PubMed1988

Exofacial regions of the glucose transporter of human erythrocytes: detection with polyclonal antibodies.

The glucose transporter of human erythrocytes is a glycoprotein of 492 amino acids with a Mr of 55,000. From hydrophobicity plots based on the transporter's amino acid sequence, it has been proposed that exofacially, there are only a segment of 34 residues and the glycosylating carbohydrate branch. To detect changes in the number of glucose transporters during metabolic regulation in intact cells, one should obtain antibodies directed to exofacial sites of the transporter. Antibodies to the purified glucose transporter (Band 4.5), intact or deglycosylated with endoglycosidase F, were raised in rabbits. These antibodies, when purified by column chromatography on protein A-Sepharose and by adsorption onto erythrocyte membranes, cross-reacted with the glycosylated glucose transporter on Western blots. The reactivity of the polyclonal antibodies with intact cells was tested by incubating these cells with the antibody, followed by a centrifugation and a subsequent reaction with 125I-labelled goat-antirabbit immunoglobulin G. Intact human erythrocytes reacted positively with the anti-Band 4.5 antibodies but not with nonimmune sera. Reaction with human erythrocytes was about 10 times greater than with pig erythrocytes, which lack glucose transporters. The reaction with intact cells was not due to contamination with broken cells since under the conditions used, broken (freeze-thawed) cells or membranes did not sediment. Reaction with human erythrocyte membranes was more than fivefold higher than with pig erythrocyte membranes. Rat L6 muscle cells reacted with anti-Band 4.5 antibodies; there were about 10 times more binding sites in any one cell in L6 cells than in human erythrocytes, roughly paralleling their relative content of glucose transporters.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

Stimulation of Na+/H+ exchange by insulin and phorbol ester during differentiation of 3T3-L1 cells. Relation to hexose uptake.

Acute exposure of 3T3-L1 undifferentiated fibroblasts to insulin or 4 beta-phorbol-12,13-dibutyrate (PDB) produced a moderate but significant stimulation of hexose transport (100% stimulation). In differentiated 3T3-L1 adipocytes, stimulation by insulin increased significantly (to 340%), while that by PDB remained at 130%. Total protein kinase C activity was 3-fold higher in 3T3-L1 fibroblast than adipocyte homogenates. PDB, but not insulin, induced migration of protein kinase C from the cytosol to the membrane, in both fibroblasts and adipocytes. Moreover, the hormone increased by 15% the protein kinase C activity of the cytosol. In 3T3-L1 fibroblasts, both insulin and PDB elicited a rapid (2 min lag) cytoplasmic alkalinization, measured with the fluorescent pH indicator bis-carboxyethyl carboxyfluorescein trapped in the cytoplasm. In 3T3-L1 adipocytes, PDB but not insulin elicited the cytoplasmic alkalinization. The alkalinization was prevented by amiloride or by replacing Na+ with either N-methylglucamine+ or K+. Stimulation of hexose transport by insulin or PDB was not affected by amiloride or Na+ substitution. It is concluded that: 1) Insulin and PDB have different effects on protein kinase C activity and subcellular distribution; 2) the responses of Na+/H+ exchange and hexose transport to insulin and PDB develop independently during differentiation of 3T3-L1 cells; 3) stimulation of Na+/H+ exchange and of hexose transport occur in parallel rather than in series in 3T3-L1 cells.

Animals↗

PubMed1987

Cell swelling following recovery from acidification in C6 glioma cells: an in vitro model of postischemic brain edema.

Two consequences of cerebral ischemia are cell acidification and cytotoxic edema. To test the possibility that Na+/H+ exchange mediates acid-induced edema, we measured cytoplasmic pH (pHi) and cell volume changes in C6 glioma cells that were artificially acid-loaded using weak electrolytes. pHi was monitored fluorimetrically with 2',7'-bis-(2-carboxyethyl)-5,6-carboxyfluorescein. Upon acidification with sodium propionate, pHi dropped to 6.74 +/- 0.05 (n = 25), and then recovered to levels near the physiological value of 7.23 +/- 0.02 (n = 13). Cell volume, measured by electronic sizing, increased concomitantly by approximately 50% in sodium propionate solution. Both pHi recovery and cell swelling were Na+-dependent, amiloride-sensitive, and inhibited at pHo less than 6.0. These results demonstrate that in vitro: (1) intracellular acidification can lead to cell swelling, and (2) pHi recovery and the concomitant cell swelling are likely mediated by Na+/H+ exchange. These mechanisms may be related to postischemic cytotoxic glial edema.

Animals↗

PubMed1987

Insulin-induced translocation of glucose transporters in rat hindlimb muscles.

Insulin causes a translocation of glucose transporters from intracellular microsomes to the plasma membrane in adipocytes. To determine whether insulin has a similar effect in rat hindlimb muscles, we used glucose-inhibitable cytochalasin B binding to estimate the number of glucose transporters in membrane fractions from insulinized and control muscles. Insulin treatment caused an approx. 2-fold increase in cytochalasin B-binding sites in a plasma membrane fraction and an approx. 70% decrease in cytochalasin B-binding sites in an intracellular membrane fraction. In order to detect this effect of insulin, it was necessary to develop a procedure for isolating a plasma membrane fraction and an intracellular membrane fraction that were not contaminated with sarcoplasmic reticulum. Our results show that, as in adipocytes, insulin stimulates translocation of glucose transporters from an intracellular membrane pool to the plasma membrane in hindlimb skeletal muscles.

Animals↗

PubMed1987

Possible mechanism for cerebral oedema in diabetic ketoacidosis.

This hypothesis, presented to explain the cerebral oedema that sometimes occurs during treatment of diabetic ketoacidosis (DKA), is based on activation of the Na+/H+ exchanger, a ubiquitous plasma-membrane transport system that functions in the regulation of cytoplasmic pH. Experimental acidification of the cytoplasm with weak organic acids activates the exchanger and, in the presence of extracellular Na+, leads to cell swelling. This swelling is osmotic, secondary to a net gain in Na+ and the anion of the weak organic acid. In DKA, cytoplasmic acidification results from high levels of circulating weak organic acids (ketoacids and free fatty acids) and activation of Na+/H+ exchange would similarly be expected. Conditions during conventional treatment of DKA should favour even greater activation of the exchanger and additional cell swelling would be predicted. The hypothesis is consistent with the clinical observation that clinically apparent cerebral oedema occurs with improvement in the patient's acid-base status rather than at the peak of the ketoacidosis.

Acid-Base Imbalance↗

PubMed1987

Cytoplasmic Ca2+ during differentiation of 3T3-L1 adipocytes. Effect of insulin and relation to glucose transport.

The cytoplasmic concentration of ionized Ca2+ [( Ca2+]i) was determined in 3T3-L1 cells during their differentiation from fibroblasts to adipocytes, suspended and loaded with the fluorescent Ca2+ indicators quin2 or indo-1. In undifferentiated fibroblasts, as well as in differentiated adipocytes up to day 9, [Ca2+]i was steady around 170 nM, and it increased significantly only in old adipocytes (day 12). During differentiation, stimulation of glucose uptake by insulin increased from a few percent to severalfold. Stimulation of uptake was already apparent after 10 min of addition of the hormone, and 10 nM insulin produced maximal stimulation in 30 min. Insulin (10(-6) M) added to quin2- or indo-1-loaded, suspended adipocytes had no detectable effect on [Ca2+]i for at least 10 min. In contrast, addition of the general anesthetic halothane increased [Ca2+]i from 172 to 251 nM in 3 min. In EGTA solution, the Ca2+ ionophore ionomycin elicited release of Ca2+ from intracellular stores that resulted in a transient increase in [Ca2+]i. A smaller but measurable Ca2+ release from intracellular stores (increasing [Ca2+]i by 20 nM) resulted upon addition of 20 micrograms/ml phosphatidic acid. In contrast, insulin did not produce any detectable release of Ca2+ from intracellular stores. Incubation of 3T3-L1 adipocytes with insulin in the presence of EGTA (the latter in excess over the Ca2+ concentration of the medium) did not prevent the stimulation of hexose uptake by the hormone, indicating that extracellular Ca2+ does not play a role in the insulin response. Furthermore, incubation of cells with quin2/AM in EGTA medium during exposure to insulin did not prevent stimulation of hexose uptake. Under these conditions it is demonstrated that intracellular quin2 suffices to chelate cytoplasmic Ca2+ even if releasable Ca2+ from intracellular stores were to pour into the cytoplasm. Thus, quin2 effectively lowers [Ca2+]i without impairing insulin action. It is concluded that insulin does not produce changes in [Ca2+]i and that chelating intracellular Ca2+ does not prevent stimulation of hexose uptake by insulin. These results suggest that it is unlikely that changes in [Ca2+]i may play a role in the transduction of information in insulin stimulation of glucose uptake in 3T3-L1 adipocytes.

Adipose Tissue↗

PubMed1987

Anaesthetic-induced increase in ionised calcium in blood mononuclear cells from malignant hyperthermia patients.

The cytoplasmic concentration of ionised calcium, [Ca2+]i, is believed to be altered by agents that induce a malignant hyperthermia (MH) crisis in susceptible individuals. MH patients were identified by the halothane and halothane/caffeine contracture tests done in isolated muscle biopsy specimens. [Ca2+]i was measured in isolated peripheral blood mononuclear cells from MH patients and controls by means of the fluorescent calcium ion indicator quin2. In the absence of halothane there was no significant difference in [Ca2+]i in cells from normal and MH patients. Addition of halothane (4 microliter/ml) significantly increased [Ca2+]i in cells from MH patients but not in controls. The halothane-induced increase in [Ca2+]i required extracellular calcium ions. This is the first evidence of the mechanism of action of halothane in cells of MH patients; the differential effect of halothane on [Ca2+]i might constitute the basis for a non-invasive screening test for MH.

Aminoquinolines↗

PubMed1987

Distribution of glucose transporters and insulin receptors in the plasma membrane and transverse tubules of skeletal muscle.

The distribution of glucose transporters and of insulin receptors on the surface membranes of skeletal muscle was studied, using isolated plasma membranes and transverse tubule preparations. (i) Plasma membranes from rabbit skeletal muscle were prepared according to Seiler and Fleischer (1982, J. Biol. Chem. 257, 13862-13871), and transverse tubules from rabbit skeletal muscle were prepared according to Rosemblatt et al. (1981, J. Biol. Chem. 256, 8140-8148) as modified by Hidalgo et al. (1983, J. Biol. Chem. 258, 13937-13945). The membranes were identified by the abundance of nitrendipine receptors in the transverse tubules, and their relative absence from the plasma membranes. (ii) Plasma membranes and transverse tubules were also isolated from rat skeletal muscle, according to a novel procedure that isolates both fractions from the same common homogenate. (iii) Glucose transporters were detected by D-glucose protectable binding of the specific inhibitor [3H]cytochalasin B, and insulin receptors were detected by saturable binding of 125I-insulin. The concentration of glucose transporters was about threefold (rabbit) or fivefold (rat) higher in the transverse tubule membrane compared to the plasma membrane, whereas the insulin receptor concentration was about the same in both membranes. These results indicate that the glucose transporters on the surface of the muscle are preferentially segregated to the transverse tubules, and this poses interesting consequences on the functional response of glucose transport to insulin in skeletal muscle.

Animals↗

PubMed1987

Protein kinase C is not required for insulin stimulation of hexose uptake in muscle cells in culture.

The L6 skeletal muscle cell line has been identified as a suitable model to study the action of insulin on glucose uptake in muscle [Klip, Li & Logan (1984) Am. J. Physiol. 247, E291-E296]. The signals that transfer information from occupied insulin receptors to glucose transporters remain unknown. Here we report that activation of protein kinase C by exogenous phorbol esters results in stimulation of glucose uptake. Protein C kinase activity was induced to migrate from the cytosolic fraction to the microsomal fraction after 40 min of exposure of intact cells to 4 beta-phorbol 12,13-dibutyrate. In contrast, incubation with insulin did not alter the subcellular distribution of the kinase. Prolonged preincubation of L6 cells with phorbol esters resulted in depletion of kinase C activity, whereas neither the basal rate of glucose uptake nor its stimulation by insulin were affected. This suggests that protein kinase C is expressed in L6 cells, and that insulin stimulation of hexose transport does not involve protein kinase C.

Cell Line↗

PubMed1987

Selective increase in cytoplasmic calcium by anesthetic in lymphocytes from malignant hyperthermia-susceptible pigs.

Anesthetic-induced malignant hyperthermia in pigs and humans is characterized by muscle rigidity and rapid, often fatal, increases in body temperature. A defect in Ca2+ homeostasis has been suspected as underlying the disease, based on the preventive effect of dantrolene sodium, an agent thought to reduce Ca2+ levels in the cytoplasm. We describe here direct measurements of cytoplasmic ionized Ca2+ levels in lymphocytes from seven normal and 12 malignant hyperthermia-susceptible pigs, using the fluorescent indicator quin2. No differences in the concentration of cytoplasmic ionized Ca2+ were found in cells from malignant hyperthermia-susceptible pigs (160 +/- 10 nM) relative to the controls (150 +/- 10 nM). However, addition of halothane in vitro caused a significant increase (to 270 +/- 30 nM) in lymphocytes from malignant hyperthermia-susceptible pigs, but not from normal pigs (180 +/- 10 nM). The halothane-mediated increase in cytoplasmic ionized Ca2+ required extracellular Ca2+. It is suggested that general anesthetics such as halothane increase the permeability of the cell surface to Ca2+, and that this increase may, on its own or indirectly, increase the cytoplasmic level of ionized Ca2+ during a malignant hyperthermia crisis. The detection of a halothane-dependent increase in cytoplasmic ionized Ca2+ selectively in malignant hyperthermia-susceptible pigs could be the basis for a noninvasive test for malignant hyperthermia.

Aminoquinolines↗

PubMed1986

Insulin stimulation of glucose uptake and the transmembrane potential of muscle cells in culture.

The membrane potential of L6 muscle cells was measured with the fluorescent dye bis-oxonol. Hyperpolarizations of up to 15 mV were caused by gramicidin (in N-methyl-D-glucamine+ medium), or by monensin or ionomycin. Depolarization was achieved with gramicidin (in Na+ medium), or with K+. Insulin did not change the resting membrane potential of -70 mV, yet it effectively stimulated 2-deoxy-D-glucose uptake. Conditions that hyperpolarize the cells did not alter the basal rate of hexose uptake. Moreover, insulin was still capable of stimulating hexose uptake in depolarized cells. It is concluded that modulation of the membrane potential is probably not a signalling event in insulin stimulation of hexose uptake.

Biological Transport↗

PubMed1986

Voltage-dependent increase in ionized cytoplasmic calcium in the L6H9 muscle cell line detected with quin2.

The concentration of free Ca2+ ions in the cytoplasm ([Ca2+]i) is a key parameter in the function of muscle cells. This study describes the effect of membrane depolarization on [Ca2+]i in differentiating cells of the L6H9 line of rat skeletal muscle. [Ca2+]i was assessed using the fluorescent indicator quin2. In the presence of 1 mM extracellular Ca2+, [Ca2+]i averaged 250 nM. Replacement of extracellular Na+ with K+ resulted in cellular depolarization from -64 to -20 mV, measured with a fluorescent oxonol indicator. Depolarized cells showed a significant increase in [Ca2+]i, from 250 to 390 nM. The increase was prevented by nifedipine (5 microM) and was in great part dependent on the presence of extracellular Ca2+. A residual significant increase in [Ca2+]i was observed upon depolarization in Ca2+-free medium; this rise may be attributed to Ca2+ release from intracellular organelles. In the presence of extracellular Ca2+, replacement of extracellular Na+ by N-methylglucamine+ did not depolarize the cells, yet resulted in a significant increase in [Ca2+]i. This rise may be ascribed to inhibition or reversal of Na+/Ca2+ exchange activity due to the absence of extracellular Na+. The data are consistent with the presence of voltage-sensitive Ca2+ channels and Na+/Ca2+ antiporters at the cell surface, and of mechanisms of voltage-sensitive Ca2+ release from intracellular organelles.

Animals↗

PubMed1986

Regulation of amino acid uptake by phorbol esters and hypertonic solutions in rat thymocytes.

Growth factors, mitogens, and malignant transformation can alter the rate of amino acid uptake in mammalian cells. It has been suggested that the effects of these stimuli on proliferation are mediated by activation of Na+/H+ exchange. In lymphocytes, Na+/H+ exchange can also be activated by phorbol esters and by hypertonic media. To determine the relationship between the cation antiport and amino acid transport, we tested the effects of these agents on the uptake of alpha-aminoisobutyric acid (AIB), methyl-AIB, proline, and leucine in rat thymocytes. Both 12-O-tetradecanoylphorbol-13-acetate (TPA) and hypertonicity stimulated amino acid uptake through system A (AIB, proline, and methyl-AIB). In addition, TPA, but not hypertonicity, also elevated leucine uptake. The stimulation of the Na+ -dependent system A was not due to an increased inward electrochemical Na+ gradient. The effects of TPA and hypertonic treatment were not identical: Stimulation of AIB uptake by TPA was observed within minutes, whereas at least 1 hr was required for the effect of hypertonicity to become noticeable. Moreover, stimulation by hypertonicity but not that by TPA, was partially inhibited by cycloheximide, suggesting a role of protein synthesis. That stimulation of Na+/H+ exchange does not mediate the effects on amino acid transport is suggested by two findings: 1) the stimulation of AIB uptake was not prevented by concentrations of amiloride or of 5-(N,N-disubstituted) amiloride analogs that completely inhibit the Na+/H+ antiport and 2) conditions that mimic the effect of the antiport, namely, increasing [Na+]i or raising pHi failed to stimulate amino acid uptake. Thus, in lymphocytes, activation of Na+/H+ exchange and stimulation of amino acid transport are not casually related.

Amino Acids↗

PubMed1986

Chemical and genetic comparison of the glucose and nucleoside transporters.

Glucose and nucleoside uptake into human red cells occurs through protein(s) which copurify in a complex, known as band 4.5 of relative mass (Mr) 66,000 to 50,000. The specific inhibitor of glucose transport, [3H]cytochalasin B, and the specific inhibitor of nucleoside transport, [3H]nitrobenzylthioribofuranosylpurine ([3H]NBMPR), incorporate covalently into component(s) of band 4.5 upon irradiation with ultraviolet light. Both photolabelled components are shown to be glycoproteins, since their migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is increased after treatment of photolabelled band 4.5 with endoglycosidase F. Peptide maps of the photolabelled components were compared. Red cell membranes were photolabelled with either [3H]cytochalasin B or [3H]NBMPR and subjected to SDS-PAGE. The region containing band 4.5 was cut and transferred to a second SDS-PAGE system and exposed to either papain or Staphylococcus aureus V8 protease. Papain (5 micrograms) completely cleaved band 4.5 and produced fragments of Mr 33,000, 26,000, 21,000, 15,000, and 12,500. Of these, the 21,000 fragment was the most conspicuous and it retained the label of [3H]cytochalasin B; the 33,000 fragment retained the label of [3H]NBMPR. The V8 protease (0.75 microgram) completely cleaved band 4.5 and produced fragments of Mr 35,000, 28,000, 22,000, 16,000, 13,500, and 9,000. The 28,000 fragment retained the label of [3H]cytochalasin B. The label of [3H]NBMPR was distributed along the gel in several regions comprising the 35,000, 28,000, and 16,000 fragments. Longer treatment with the V8 protease did not alter the position of the 28,000 [3H]cytochalasin B labelled peak, but completely abolished the [3H]NBMPR labelled peaks. Genetic segregation of the glucose and nucleoside transporters was determined in a lymphoma cell line. A mutant (14T- g) of S49 cells was selected which had lost the capacity to transport thymidine or to bind NBMPR. Uptake of either 2-deoxyglucose or 3-O-methylglucose, inhibitable by cytochalasin B, was not impaired in this mutant. It is concluded that the nucleoside and glucose transporters are glycoprotein components of band 4.5, which are differentiated by peptide map analysis. Further, a lymphoblast mutant was isolated which had lost the nucleoside transport function but retained the glucose transport function.

Carrier Proteins↗

PubMed1986

Changes in cytoplasmic free calcium caused by halothane. Role of the plasma membrane and intracellular Ca2+ stores.

Malignant hyperthermia is a muscle disease characterized by an abnormal response to anaesthetics, stress, and exercise. It is typified by muscle contracture and a dramatic elevation in body temperature. A defect in the regulation of the concentration of cytoplasmic free calcium, [Ca2]i, is thought to underlie this disease, but the actual [Ca2+]i was not measurable until recently. We have shown that the anaesthetic halothane increases [Ca2+]i in isolated lymphocytes from malignant hyperthermia-susceptible humans and pigs but not in the normal counterparts. In this report we extend these observations to a larger number of cases and analyze the molecular mechanisms responsible for the increase. The halothane-mediated rise in [Ca2+]i required external Ca2+ and was prevented by nifedipine, an inhibitor of the voltage-sensitive Ca2+ channels of the cell membrane. In addition, the effect of halothane on the releasable Ca2+ from intracellular stores was determined by measuring the size of the releasable pool before and after addition of the anaesthetic. After addition of halothane, about 73% of this Ca2+ pool was still available for release by the Ca2+ ionophore ionomycin in cells from normal humans and pigs. In contrast, only about 45% of the free Ca2+ in intracellular stores was left after treatment with halothane in cells from malignant hyperthermia-susceptible humans and swine. These results indicate that halothane acts both at the cell membrane and at intracellular organelles, and that this action results in a net increase in [Ca2+]i in malignant hyperthermia, but not in normal cells. The action at the cell membrane appears to be on the voltage-sensitive Ca2+ channels.(ABSTRACT TRUNCATED AT 250 WORDS)

Aminoquinolines↗

PubMed1986

Insulin-induced cytoplasmic alkalinization and glucose transport in muscle cells.

Insulin stimulates glucose uptake into muscle within minutes, preceding stimulation of glycolysis. Signals involved in stimulation of glycolysis include cytoplasmic alkalinization and specific intracellular proteolytic products. In contrast, the signals that mediate stimulation of glucose transport remain unknown. Here we explore whether the insulin-induced cytoplasmic alkalinization is an early event that precedes activation of sugar uptake, whether such alkalinization is causally related to stimulation of sugar uptake, and whether proteolytic activity mediates stimulation of hexose transport. Cytoplasmic pH (pHi) was measured in suspended skeletal muscle cells of the L6H9 line with the intracellularly trapped fluorescent pH indicator bis(carboxyethyl)carboxy fluorescein. At 37 degrees C, insulin (1 X 10(-7) M) produced an increase in pHi of 0.11 units in 10 min. This increase became apparent 2 min after addition of the hormone, and maximal elevation of pHi was observed after 10 min, remaining elevated for up to 60 min. Removal of the hormone with anti-insulin antiserum did not reverse pHi back to the resting level. The alkalinization was prevented by amiloride, by 5-(N,N'-disubstituted)amiloride analogues, and by isosmotic replacement of Na+ with N-methylglucamine+ or choline+. This suggests that insulin activates Na+-H+ exchange. In contrast, stimulation of 2-deoxy-D-glucose transport by insulin was not affected by replacement of external Na+ or by addition of amiloride. Monensin, an exogenous Na+-H+ exchanger, did not stimulate sugar transport even though it increased pHi. Proteinase inhibitors that block hormonal stimulation of glycolysis were ineffective in preventing stimulation of 2-deoxy-D-glucose transport by insulin.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride↗

PubMed1985

The free cytoplasmic Ca2+ levels in Duchenne muscular dystrophy lymphocytes.

An increased cellular Ca2+ content has been associated with Duchenne muscular dystrophy (DMD). However, estimates of the free cytoplasmic Ca2+ concentration ([Ca2+]i) in cells of DMD patients were not available. We compared the [Ca2+]i levels of normal and DMD peripheral blood lymphocytes and Epstein-Barr virus-transformed lymphoblasts using the novel probe, quin 2, an internally trapped fluorescent indicator. The [Ca2+]i levels of normal and DMD cells were not significantly different.

Calcium↗