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M Lieberman

Publications and source records attributed to M Lieberman.

At least 55 records · Page 3Linked to original sources
PubMed1994

Calcium depletion and repletion in cultured chick heart muscle cells.

Calcium-free incubation followed by exposure to calcium damages naturally occurring cardiac muscle preparations irreversibly. Whether the observed calcium overload during calcium repletion is a primary cause for, or a secondary consequence of, sarcolemmal disruption and cell injury is controversial. We used cultured embryonic chicken heart muscle cells to correlate ionic, metabolic, and ultrastructural changes during calcium depletion (0Ca, 1 mM EGTA) and repletion. After 10 min of calcium depletion, intracellular Na increased four-fold above control levels, intracellular K decreased by 26%, total cell Ca decreased by 81%, and cytosolic ionized Ca concentration decreased by 87%. Within 10 min of subsequent calcium repletion, total cell Ca transiently increased to four-fold above control, cytosolic ionized Ca concentration transiently increased to twice control, and both Na and K returned toward control levels; by 3 h of calcium repletion, physiological cation (Na, K, Ca) contents were restored and adenine nucleotide contents were normal. Long-term (i.e. 120 min) calcium depletion did not significantly reduce cell ATP levels, but increased adenine nucleotide turnover as indicated by adenosine and lactate release; after 60 min of subsequent calcium repletion, ionic and metabolic parameters were returned to control levels. During calcium depletion (both short- and long-term) and subsequent repletion, no ultrastructural changes occurred. When Mg was also removed during calcium depletion, the ionic changes during depletion and subsequent repletion were enhanced. When 10 microM CCCP was present during calcium depletion and repletion, cytosolic ionized Ca concentration increased to six-fold above control with no increase in total cell Ca content, suggesting that the increased Ca is buffered, in part, by mitochondria. These results indicate that an increase in Ca per se, occurring when high energy phosphate levels and/or cellular Ca buffering capacity are maintained, does not seem to be associated with irreversible cell injury.

Animals↗
PubMed1994

A novel Cl- conductance in cultured chick cardiac myocytes: role of intracellular Ca2+ and cAMP.

Cl- conductance in cultured embryonic chick cardiac myocytes was characterized using whole-cell patch clamp techniques. Following elimination of cation currents in Na(+)- and K(+)-free internal and external solutions, the basal whole-cell current was predominantly a Cl- current. Cl(-)-sensitive current (ICl) was defined as the difference between the whole-cell currents recorded in normal and low [Cl-]o when measured in the same cell. The whole-cell current in the absence or presence of 10 microM cAMP was time independent, displayed outward rectification with the pipette [Cl-] < 40 mM, and was not saturated with a physiological Cl- gradient. The Cl- current was also activated by 1 microM forskolin and inhibited by 0.3 mM anthracene-9-carboxylic acid (9-AC). Forskolin was less effective than cAMP (internal dialysis) in activating the Cl- current. The cAMP- or forskolin-activated and basal Cl- current were reasonably fit by the Goldman-Hodgkin-Katz equation. The calculated PCl in the presence of cAMP was increased by five- to sixfold over the basal level. In the presence of 5 mM EGTA to decrease free [Ca2+]i, the whole-cell current could not be stimulated by cAMP, forskolin or IBMX (0.1 mM). These data suggest that cultured chick cardiac myocytes have a low basal Cl- conductance, which, as in some mammalian cardiac ventricular myocytes, can be activated by cAMP. However, this study shows that the activation process requires physiological free [Ca2+]i.

Animals↗
PubMed1994

Mitochondrial localization and characterization of 99Tc-SESTAMIBI in heart cells by electron probe X-ray microanalysis and 99Tc-NMR spectroscopy.

As the development of targeted intracellular magnetic resonance contrast agents proceeds, techniques for the quantitative analysis of the subcellular compartmentation and characterization of metallopharmaceuticals must also advance. To this end, the subcellular distribution and chemical state of hexakis (2-methoxyisobutyl isonitrile) technetium-99 (99Tc-SESTAMIBI), the ground state of the organotechnetium radiopharmaceutical used for the noninvasive evaluation of myocardial perfusion and viability by scintigraphy, has been determined by a novel application of electron probe X-ray microanalysis (EPXMA) and 99Tc-NMR spectroscopy. In cryopreserved cultured chick heart cells equilibrated in 36 microM 99Tc-SESTAMIBI, EPXMA imaging of mitochondria yielded a respiratory uncoupler-sensitive characteristic 99Tc X-ray peak representing 32.0 +/- 2.9 nmoles Tc/mg dry weight, while EPXMA of cytoplasm or nucleus showed no peak significantly greater than the threshold detectability limit of approximately 1 nmole/mg dry weight. Technetium-99 NMR spectroscopy of heart cells equilibrated with 99Tc-SESTAMIBI showed a single peak at -45.5 ppm with no evidence of significant line broadening or chemical shift compared to aqueous chemical standards, indicating that the majority of the complex exists unbound within the mitochondrial matrix. These data quantitatively demonstrate the localization of this lipophilic cationic organometallic complex within mitochondria in situ, consistent with a sequestration mechanism dependent on membrane potentials. Furthermore, this study establishes the general feasibility of combined EPXMA and NMR spectroscopy for the direct subcellular localization and characterization of metallopharmaceuticals, techniques that are readily applicable to MR contrast agents.

Animals↗
PubMed1994

Electrodiffusive movements of chloride ion in sodium-free solution: a possible calcium activated chloride conductance in cultured heart cells.

OBJECTIVE: The aim was to demonstrate that removal of extracellular sodium (Na+o), a manoeuvre known to increase intracellular calcium (Cai2+), can activate a chloride ion conductance in cultured embryonic chick heart cells. METHODS: Intracellular chloride activity (aiCl) and membrane potentials (Em) were simultaneously measured using chloride selective and 3 M KCl filled microelectrodes. RESULTS: In Na+o-free and K+o-free solutions, a frusemide (0.3 mM) resistant decrease in aiCl of 10 mM was recorded within 10 min, along with a 5 mV hyperpolarisation, and the calculated chloride equilibrium potential (ECl) followed the change of Em, suggesting a possibly enhanced chloride conductance. When cells were exposed to Na+o-free solution, the decrease of aiCl by approximately 10 mM was associated with an initial depolarisation, followed by a hyperpolarisation to approximately -60 mV (more negative than ECl). Subsequent removal of K+o depolarised Em to -39 mV (approximately ECl), and no further loss of aiCl occurred. Restoration of K+o caused a hyperpolarisation of Em (more negative than ECl) and a continuing decline of aiCl. Prevention of K+o induced hyperpolarisation by addition of 1 mM Ba2+ stopped the decline of aiCl (Em approximately ECl), suggesting that following Na+o removal, alteration of the driving force for chloride led to a chloride efflux via an enhanced chloride conductance. When Em depolarised to -50 mV after 3 min exposure to 0.1 mM ouabain, removal of [Cl-]o caused a further depolarisation and readdition of [Cl-]o induced an 18 mV hyperpolarisation. This chloride induced hyperpolarisation was blocked by removal of [Ca2+]o (+1 mM EGTA). CONCLUSIONS: The increase in chloride conductance observed under conditions that are known to raise Ca2+i supports the presence of a Ca2+i activated chloride conductance in cultured chick heart cells.

Animals↗
PubMed1994

Radiation leukemia virus-induced thymic lymphomas express a restricted repertoire of T-cell receptor V beta gene products.

We have investigated the phenotypic changes that take place during the process of neoplastic transformation in the thymocytes of C57BL/Ka mice infected by the radiation leukemia virus (RadLV). By the combined use of antibodies against the envelope glycoprotein gp70 of RadLV, the transformation-associated cell surface marker 1C11, and the CD3-T-cell receptor (TCR) complex, we found that in the RadLV-infected thymus, the earliest expression of viral gp70 is in 1C11hi cells; a small but significant percentage of these cells also express CD3. A first wave of viral replication, manifested by the expression of high levels of gp70 in thymocytes (over 70% positive), reaches a peak at 2 weeks; during this period, no significant changes are observed in the expression of 1C11 or CD3. The population of gp70+ cells is drastically reduced at 3 to 4 weeks after infection. However, a second cohort of gp70+ cells appears after 4 weeks, and these cells express high levels of 1C11 and TCR determinants as well. RadLV-induced lymphomas differ from normal thymocytes in their CD4 CD8 phenotype, with domination by one or more subsets. Characterization of TCR gene rearrangements in RadLV-induced lymphomas shows that most of these tumors are clonal or oligoclonal with respect to the J beta 2 TCR gene, while the J beta 1 TCR gene is rearranged in a minority (4 of 11) of lymphomas. TCR V beta repertoire analysis of 12 tumors reveals that 6 (50%) express exclusively the V beta 6 gene product, 2 (17%) are V beta 5+, and 1 (8%) each are V beta 8+ and V beta 9+. In normal C57BL/Ka mice, V beta 6 is expressed on 12%, V beta 5 is expressed on 9%, V beta 8 is expressed on 22%, and V beta 9 is expressed on 4% of TCRhi thymocytes. Thus, it appears that RadLV-induced thymic lymphomas are not randomly selected with respect to expressed TCR V beta type.

Animals↗
PubMed1994

Sepsis stimulates nonlysosomal, energy-dependent proteolysis and increases ubiquitin mRNA levels in rat skeletal muscle.

We tested the role of different intracellular proteolytic pathways in sepsis-induced muscle proteolysis. Sepsis was induced in rats by cecal ligation and puncture; controls were sham operated. Total and myofibrillar proteolysis was determined in incubated extensor digitorum longus muscles as release of tyrosine and 3-methylhistidine, respectively. Lysosomal proteolysis was assessed by using the lysosomotropic agents NH4Cl, chloroquine, leupeptin, and methylamine. Ca(2+)-dependent proteolysis was determined in the absence or presence of Ca2+ or by blocking the Ca(2+)-dependent proteases calpain I and II. Energy-dependent proteolysis was determined in muscles depleted of ATP by 2-deoxyglucose and 2.4-dinitrophenol. Muscle ubiquitin mRNA and the concentrations of free and conjugated ubiquitin were determined by Northern and Western blots, respectively, to assess the role of the ATP-ubiquitin-dependent proteolytic pathway. Total and myofibrillar protein breakdown was increased during sepsis by 50 and 440%, respectively. Lysosomal and Ca(2+)-dependent proteolysis was similar in control and septic rats. In contrast, energy-dependent total and myofibrillar protein breakdown was increased by 172% and more than fourfold, respectively, in septic muscle. Ubiquitin mRNA was increased severalfold in septic muscle. The results suggest that the increase in muscle proteolysis during sepsis is due to an increase in nonlysosomal energy-dependent protein breakdown, which may involve the ubiquitin system.

Animals↗
PubMed1993

Ion transport during hypothermia in cultured heart cells: implications for protection of the immature myocardium.

In non-adult hearts, hypothermia influences protection of the myocardium by exerting effects on specific ion transporters, thereby altering the normal balance between ion pumps and ion leaks. We studied the effects of hypothermia on individual ion transporters in cardiac myocytes to better understand how to preserve the normal ion balance at reduced temperatures, and thereby enhance myocardial protection. Cardiocytes obtained from 11 day chick embryos were cultured for 3 days, and then equilibrated in a glucose containing HEPES-TRIS buffered salt solution at 37 degrees C (pH = 7.4). The cells were incubated at 10 +/- 2 degrees C for 5 to 360 min in the absence or presence of specific ion transport inhibitors, and ion contents were assessed by atomic absorption spectrophotometry. Intracellular Na content increased from approximately 90 nmol/mg protein (control) to 2-3 times this value within 30 min, and then returned to control levels by 60 min. This increase in Na was accompanied by a small rise in total Ca (1.5 times control). Acidotic pH (6.4) and/or ethylisopropyl amiloride (100 microM), but not bumetanide (100 microM) prevented the rise in Na content, suggesting the Na/H exchanger contributed to the initial Na influx. Ouabain (1 mM), exacerbated the Na rise and prevented its recovery to control values at 10 degrees C, although Rb flux measurements revealed only a low level of Na/K ATPase activity throughout 240 min at 10 degrees C (15% of 37 degrees C activity). Calcium content rose to 10 times control values in the presence of ouabain at 37 degrees C only, consistent with a lack of significant Na/Ca exchange activity during hypothermia. In conclusion, the effects of hypothermia on ion pumps and ion leaks in embryonic heart cells are as follows: (1) a low level of Na/K ATPase activity contributes significantly to ion regulation; (2) activity of the Na/H exchanger must be attenuated to minimize Na loading; (3) slowing of the Na/Ca exchange may reduce Ca induced cell injury. We suggest that reducing Na/H exchange activity during hypothermia, using cardioplegic solutions with a slightly acidic pH or with added ethylisopropyl amiloride, may enhance the protective effects of hypothermia in non-adult hearts.

Animals↗
PubMed1993

Cardiac cell toxicity induced by 4-hydroperoxycyclophosphamide is modulated by glutathione.

OBJECTIVE: Cardiac myocytes were exposed to 4-hydroperoxycyclophosphamide (4-HC, an activated derivative of cyclophosphamide) to assess whether early ionic events are associated with the dose limiting toxicity of this chemotherapeutic agent. METHODS: Primary cultures of embryonic chick cardiac myocytes were grown to confluency and then exposed to a medium containing 4-HC. Cellular sodium, potassium, and calcium contents were measured by atomic absorption spectrophotometry and related to protein and ATP content. Pretreatment of the cultured heart cells with glutathione depleting or enhancing agents provided the basis for evaluating the involvement of glutathione in the 4-HC-induced cytotoxicity. RESULTS: Administration of 150 microM 4-HC to cardiac myocytes resulted in increases in cellular sodium and calcium contents and decreases in potassium, ATP, and protein contents. Pretreatment of cardiac myocytes with L-buthionine-SR-sulphoximine, a specific inhibitor of gamma-glutamylcysteine synthetase, depleted cellular glutathione to 12% of control and significantly reduced the minimum concentration of 4-HC causing cytotoxic changes. Conversely, elevation of cellular thiol content by the pretreatment of cardiac myocytes with glutathione monoethyl ester (but not glutathione) provided protection against 4-HC induced cytotoxicity. CONCLUSIONS: Cellular glutahione concentration can markedly influence the 4-HC induced changes in cellular ion content and ATP, which are early indicators of 4-HC induced cytotoxicity.

Adenosine Triphosphate↗
PubMed1993

A chloride current associated with swelling of cultured chick heart cells.

1. Cultured chick heart cells challenged by hyposmotic stress underwent regulatory volume decrease (RVD) that was attenuated by prior depletion of intracellular chloride. 2. During hyposmotic swelling, cell aggregates experienced an initial increase in spontaneous contractile activity followed by eventual quiescence. Conventional microelectrode studies revealed an underlying increase in spontaneous electrical activity, followed by a sustained depolarization beyond threshold. 3. Whole-cell patch clamp studies, with K+ currents blocked, indicated that exposure of cells to hyposmotic solution (NaCl reduction) resulted in a rapid osmotic swelling followed by a substantial increase in whole-cell conductance which persisted for the duration of hyposmotic exposure and was almost completely reversed on return to isosmotic bath solution. 4. For a variety of Cl- concentrations, the reversal potentials (Erev) of the measured swelling-activated current closely followed the calculated Cl- equilibrium potential (ECl) with a linear regression slope of 0.82. When estimated by the Nernst equation, the relationship between Erev and the [Cl-]i/[Cl-]o ratio fitted well with a slope of 51 mV per decade change in the concentration ratio, consistent with a Cl(-)-selective conductance. 5. The permeability ratios of this swelling-activated conductance to chloride, methanesulphonate (MSA) and aspartate (Asp) were calculated as PCl:PMSA:PASP = 1:0.36:0.02, with the ion selectivity sequence of Cl- > MSA- >> Asp-, which suggests the swelling-activated conductance is slightly permeable to other anions. 6. Application of a Cl- channel blocker, diphenylamine-2-carboxylate (DPC, 200 microM), substantially suppressed the swelling-activated current without shifting the Erev of this current. The effect of DPC was independent of membrane potential. 7. This evidence demonstrates that hyposmotic swelling of cultured chick heart cells activates a channel-mediated Cl- conductance which may be associated with the integrated response of volume-regulatory mechanisms.

Animals↗
PubMed1993

Amino acid loss during volume regulatory decrease in cultured chick heart cells.

Mechanisms of volume regulation in hyposomotically treated cultured chick heart cell preparations were studied using optical, biochemical, and nuclear magnetic resonance methods. This approach afforded the resolution of time-dependent responses that might ordinarily be obscured by the complex morphology of intact cardiac muscle preparations. In hyposmotic solutions, cells swelled to a peak volume within 3 min and slowly regulated toward original volume (regulatory volume decrease, RVD). Upon return of the cells to isosmotic solution following hyposmotic treatment, the cells shrank to a steady-state volume that was substantially less than the initial volume in control solution. A vigorous RVD could also be elicited by hyposmotic swelling under Cl(-)-free conditions. Measurement of both inorganic cation loss via atomic absorption spectroscopy and organic solute loss via 1H-nuclear magnetic resonance and high-pressure liquid chromatographic techniques revealed that the RVD observed following exposure to hyposomotic solutions was mediated in part by a substantial loss of taurine, glutamate, aspartate, and glycine as well as loss of inorganic ions (Na+,K+). The hyposmotically activated transport of amino acids was also associated with the production of glutamate and aspartate. The volume regulatory release and production of amino acids have significant implications for the metabolic and functional integrity of cardiac cells.

Amino Acids↗
PubMed1993

Microprobe analysis of Tc-MIBI in heart cells: calculation of mitochondrial membrane potential.

Hexakis (2-methoxyisobutylisonitrile) technetium-99m (99mTc-MIBI) is a gamma-emitting radiopharmaceutical probe currently in clinical use to evaluate myocardial perfusion. Biochemical and cellular pharmacological studies have suggested that Tc-MIBI, a lipophilic cation, is sequestered in mitochondria in response to transmembrane potentials. To assess directly the subcellular distribution of the probe in heart tissue, cultured chick heart cells were analyzed by electron-probe X-ray microanalysis (EPXMA) following equilibration in micromolar concentrations of carrier-added 99Tc-MIBI, the ground-state radiopharmaceutical. Quantitation of the physiological elements Na, Ca, Mg, K, S, P, and Cl was correlated with exposure to increasing concentrations of 99Tc-MIBI. EPXMA signals indicated that 99Tc-MIBI was concentrated up to 1,000 times into mitochondria in a dose-dependent fashion based on measured Tc content in the mitochondria. Inner membrane potential (delta psi) of individual mitochondria was calculated as -117 mV using the Nernst equation. Concentrations of 99Tc-MIBI > 36 microM caused a significant efflux of K and Mg from the cell, as well as an increase in Cl in the mitochondria. Comparison of cell ultrastructure with conventional electron microscopy at extracellular 99Tc-MIBI concentrations of 36-72 microM showed no changes compared with control. 99Tc-MIBI allows valuable in situ investigation of cellular bioenergetics with EPXMA by quantitation of delta psi.

Animals↗
PubMed1992

Magnesium homeostasis in cardiac cells.

Several aspects of Mg2+ homeostasis were investigated in cultured chicken heart cells using the fluorescent Mg2+ indicator, FURAPTRA. The concentration of cytosolic Mg2+ ([Mg2+]i) is 0.48 +/- 0.03 mM (n = 31). To test whether a putative Na/Mg exchange mechanism controls [Mg2+]i below electrochemical equilibrium, we manipulated the Na+ gradient and assessed the effects on [Mg2+]i. When extracellular Na+ was removed, [Mg2+]i increased; this increase was not altered in Mg-free solutions, but was attenuated in Ca-free solutions. A similar increase in [Mg2+]i, which was dependent upon extracellular Ca2+, was observed when intracellular Na+ was raised by inhibiting the Na/K pump with ouabain. These results do not provide evidence for Na/Mg exchange in heart cells, but they suggest that Ca2+ can modulate [Mg2+]i. In addition, removing extracellular Na+ caused a decrease in intracellular pH (pHi), as measured by pH-sensitive microelectrodes, and this acidification was attenuated when Ca2+ was also removed from the solution. These results suggest that Ca2+ and H+ interact intracellularly. Since changes in the Na+ gradient can also alter pHi, we questioned whether pH can modulate [Mg2+]i. pHi was manipulated by the NH4Cl prepulse method. NH4(+)-evoked changes in pHi, as measured by the fluorescent indicator BCECF, were accompanied by opposite changes in [Mg2+]i; [Mg2+]i changed by -0.16 mM/unit pH. These NH4(+)-evoked changes in [Mg2+]i were not caused by movements of Mg2+ or Ca2+ across the sarcolemma or by changes in cytosolic Ca2+. Additionally, pHi was manipulated by changing extracellular pH (pHo).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Unexpected effects of the severe combined immunodeficiency mutation on murine lymphomagenesis.

Strain C.B17 scid/scid (SCID) mice, which lack functional T and B lymphocytes, show heightened susceptibility to the induction of thymic lymphomas by x-irradiation. Susceptibility is highest in thymus-chimeric SCID-BL mice (thymectomized SCID mice bearing a C57BL thymus graft). All SCID-BL lymphomas originate in the cells of the thymic graft (C57BL type) and lack murine leukemia virus expression. Both SCID and SCID-BL lymphomas are phenotypically CD4-8+ and/or CD4+8+, but only the SCID-BL tumors express CD3. Injection of C57BL or BALB/c bone marrow into irradiated SCID-BL mice prevents lymphoma development, but SCID marrow is completely ineffective. The results suggest that the scid condition enhances the activity of a putative lymphomagenic agent induced in the bone marrow by x-irradiation and that C57BL thymic cells are highly sensitive targets. Moreover, the failure of SCID bone marrow to protect against lymphomagenesis vs. the efficacy of marrow from immunocompetent donors points to involvement of T or B lineage cells in this process.

Animals↗
PubMed1992

Differentiation of CD3-4-8- thymocytes in short-term thymic stromal cell culture.

We have investigated the ability of a heterogeneous thymic stromal cell (HTSC) culture system to promote in vitro differentiation of CD3-4-8- thymocytes. Culture of purified murine CD3-4-8- thymocytes on HTSC for 1 d resulted in the appearance of CD4+8+ cells, which did not occur when the sorted cells were maintained in medium alone. It is remarkable that when the culture period was extended to 2 d, CD3-4-8- progenitors differentiated further to CD4+8- and CD4-8+ cells, which also expressed high levels of TCR-CD3. This rapid differentiation on stroma in vitro appears to outpace parallel development in vivo. The differentiation potential of a subset of CD3-4-8- thymocytes that express high levels of a marker of normal and neoplastic thymic progenitors, the 1C11 antigen, was examined next. 1C11hiCD3-4-8- cells also gave rise to CD4-8+ and CD4+8+ populations after 1 d of culture on HTSC. Extending the culture period to 2 d resulted in a significant percentage of CD3-expressing cells that were CD4+8+, CD4+8- and CD4-8+ cells. These results suggest that in the in vitro HTSC culture system, various subsets of immature thymocytes can differentiate into all the mature phenotypes of cells normally found in the adult mouse thymus. This may provide a novel and rapid assay for thymic progenitors.

Animals↗
PubMed1992

Quick-freezing of cultured cardiac cells in situ with special attention to the mitochondrial ultrastructure.

A new method has been developed which allows quick-freezing in situ of primary, cardiac cell cultures grown to confluence on gas-permeable membranes (Petriperm dishes). Small pieces of the growth substratum, with rhythmically beating myocardial cells, were slam-frozen, without cryoprotectants, against the surface of a helium-cooled copper block at approximately 16 K. The quality of the cellular cryopreservation, as judged by ultrastructural criteria, was studied in freeze-substituted specimens processed for transmission electron microscopy. The ultrastructure of cryofixed cardiac cells was compared with that of unfrozen, chemically fixed samples. The severity of cryodistortions increased progressively with increasing distance from the point of first impact. Of particular interest were the dramatic alterations of the mitochondrial ultrastructure. The concept that the reticular and the outer mitochondrial membranes are intimately and strongly associated was clearly demonstrated. Optimally frozen material revealed cryopreserved ultrastructure of high quality. The method described appears to offer an ideal model system for correlating the information gained by phase-contrast microscopy of living cell cultures with the ultrastructure of the same samples fixed in situ by chemical or physical techniques. Cryofixation would be particularly useful for studying dynamic cellular processes associated with physiological and pathophysiological conditions, e.g. metabolic inhibition, anoxia and substrate deprivation.

Animals↗
PubMed1992

Intracellular pH modulates cytosolic free magnesium in cultured chicken heart cells.

To assess the role of pH in cellular Mg homeostasis, cytosolic pH (pHi) was manipulated by the NH4Cl prepulse technique; pHi, cytosolic Mg2+ (Mgi), and cytosolic Ca2+ (Cai) were measured fluorometrically in single cultured embryonic chicken heart cells loaded with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), FURAPTRA, and fura-2, respectively. The basal values obtained were as follows: pHi = 7.21 +/- 0.10 (n = 7), [Mg]i = 0.51 +/- 0.08 mM (n = 9), [Ca]i = 126 +/- 15 nM (n = 7). When cells were perfused with 10 mM NH4Cl solution for 5 min, a transient alkalinization (0.53 U) of the cytosol was accompanied by a transient decrease (0.12 mM) in [Mg]i and a transient increase (59 nM) in [Ca]i; these changes approached control levels within 5 min. Upon removal of NH4Cl, a transient acidification (0.89 U) of the cytosol was accompanied by a transient increase (0.10 mM) in [Mg]i and a transient increase (125 nM) in [Ca]i; again, these changes returned toward control levels within 5 min. No significant changes in total cell Mg or Ca were observed during these manipulations. NH4Cl-evoked changes in [Mg]i were not altered significantly by either Mg-free or Ca-free conditions. Changes in [Mg]i were inversely correlated with changes in pHi and were not secondary to changes in [Ca]i. The results suggest that pHi modulates Mgi, probably by affecting cytosolic Mg binding and/or the transport of Mg across subcellular organelles.

Ammonium Chloride↗