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P K Lauf

Publications and source records attributed to P K Lauf.

At least 19 recordsLinked to original sources
PubMed2000

K-Cl cotransport in vascular smooth muscle and erythrocytes: possible implication in vasodilation.

K-Cl cotransport, the electroneutral-coupled movement of K and Cl ions, plays an important role in regulatory volume decrease. We recently reported that nitrite, a nitric oxide derivative possessing potent vasodilation properties, stimulates K-Cl cotransport in low-K sheep red blood cells (LK SRBCs). We hypothesized that activation of vascular smooth muscle (VSM) K-Cl cotransport by vasodilators decreases VSM tension. Here we tested this hypothesis by comparing the effects of commonly used vasodilators, hydralazine (HYZ), sodium nitroprusside, isosorbide mononitrate, and pentaerythritol, on K-Cl cotransport in LK SRBCs and in primary cultures of rat VSM cells (VSMCs) and of HYZ-induced K-Cl cotransport activation on relaxation of isolated porcine coronary rings. K-Cl cotransport was measured as the Cl-dependent K efflux or Rb influx in the presence and absence of inhibitors for other K/Rb transport pathways. All vasodilators activated K-Cl cotransport in LK SRBCs and HYZ in VSMCs, and this activation was inhibited by calyculin and genistein, two inhibitors of K-Cl cotransport. KT-5823, a specific inhibitor of protein kinase G, abolished the sodium nitroprusside-stimulated K-Cl cotransport in LK SRBCs, suggesting involvement of the cGMP pathway in K-Cl cotransport activation. Hydralazine, in a dose-dependent manner, and sodium nitroprusside relaxed (independently of the endothelium) precontracted arteries when only K-Cl cotransport was operating and other pathways for K/Rb transport, including the Ca-activated K channel, were inhibited. Our findings suggest that K-Cl cotransport may be involved in vasodilation.

Alkaloids↗
PubMed1998

Role of nitrite, a nitric oxide derivative, in K-Cl cotransport activation of low-potassium sheep red blood cells.

K-Cl cotransport (COT) is the coupled movement of K and Cl, present in most cells, associated with regulatory volume decrease, susceptible to oxidation and functionally overexpressed in sickle cell anemia. The aim of this study was to characterize the effect of the oxidant nitrite (NO2-) on K-Cl COT. NO2- is a stable metabolic end product of the short-lived highly reactive free radical nitric oxide (NO), an oxidant and modulator of ion channels, and a vasodilator. In some systems, the response to NO2- is identical to that of NO. We hypothesized that NO2- activates K-Cl COT. Low potassium (LK) sheep red blood cells (SRBCs) were used as a model. The effect of various concentrations (10(-6) to 10(-1) m) of NaNO2 was studied on K efflux in hypotonic Cl and NO3 media, Cl-dependent K efflux (K-Cl COT), glutathione (GSH), and methemoglobin (MetHb) formation. In support of our hypothesis, K efflux and K-Cl COT were stimulated by increasing concentrations of NaNO2. Stimulation of K efflux was dependent upon external Cl and exhibited a lag phase, consistent with activation of K-Cl COT through a regulatory mechanism. Exposure of LK SRBCs to NaNO2 decreased GSH, an effect characteristic of a thiol-oxidizing agent, and induced MetHb formation. K-Cl COT activity was positively correlated with Methb formation. N-ethyl-maleimide (NEM), a potent activator of K-Cl COT, was used to assess the mechanism of NO2- action. The results suggest that NEM and NO2- utilize at least one common pathway for K-Cl COT activation. Since NaNO2 is also a well known vasodilator, the present findings suggest a role of K-Cl COT in vasodilation.

Animals↗
PubMed1998

Functional evidence for a pH sensor of erythrocyte K-Cl cotransport through inhibition by internal protons and diethylpyrocarbonate.

The sidedness of proton modulation of K-Cl cotransport (K-Cl COT) was studied in low K sheep red blood cells stripped of cellular Mg, Mgi, at alkaline medium pH, pHo, by the divalent ionophore A23187 and a chelator. This procedure activates K-Cl COT, presumably, by inhibition of MgATP-dependent kinases. Ouabain-resistant K efflux and Rb influx were measured in Cl or NO3 either at variable pHi and fixed pHo, or vice versa, in erythrocytes pH- and volume-clamped with the anion exchange inhibitor 4,4'-diisothiocyanato-2,2'-disulfonic stilbene (DIDS) and sucrose. Between pHi 9 and 6, and at constant pHo 9, K effluxes decreased hyperbolically in Cl and linearly in NO3 whereas Rb influxes fell almost linearly in Cl and asymptotically in NO3. Thus, saturation of outward and inward K-Cl COT, the calculated difference of the fluxes in Cl and NO3, occurred at slightly different pHi values. Hill plots revealed pKa values of 6.5 and 7.0, and Hill coefficients of > 1 for outward and inward K-Cl COT, respectively. Raising pHo from 6 to 9 at fixed pHi slightly increased K and Rb fluxes in both Cl or NO3, but not K-Cl COT. The histidine reagent diethylpyrocarbonate (DEPC) inhibited low Mgi-activated K-Cl COT at approximately 4 mM, an effect partially reversible by subsequent treatment with hydroxylamine. It is concluded that protons inhibit erythrocyte K-Cl COT through internal histidine(s) which may be part of a pH sensor.

Animals↗
PubMed1997

Alkaline pH and internal calcium increase Na+ and K+ effluxes in LK sheep red blood cells in Cl--free solutions.

We examined the effects of pH, internal ionized Ca (Ca2+i), cellular ATP, external divalent cations and quinine on Cl-independent ouabain-resistant K+ efflux in volume-clamped sheep red blood cells (SRBCs) of normal high (HK) and low (LK) intracellular K+ phenotypes. In LK SRBCs the K+ efflux was higher at pH 9.0 (350%) than at pHs 7. 4 and 6.5, and was inhibited by external divalent cations, quinine, and cellular ATP depletion. The above findings suggest that the increased K+ efflux at alkaline pH is due to the opening of ion channels or specific transporters in the cell membrane. In addition, K+ efflux was activated (100%) when Ca2+i was increased (+A23187, +Ca2+o) into the microm range. However, in comparison to human red blood cells, the Ca2+i-induced increase in K+ efflux in LK SRBCs was fourfold smaller and insensitive to quinine and charybdotoxin. The Na+ efflux was also higher at pH 9.0 than at pH 7.4, and activated (about 40%) by increasing Ca2+i. In contrast, in HK SRBCs the K+ efflux at pH 9.0 was neither inhibited by quinine nor activated by Ca2+i. These studies suggest the presence in LK SRBCs, of at least two pathways for Cl--independent K+ and Na+ transport, of which one is unmasked by alkalinization, and the other by a rise in Ca2+i.

Adenosine Triphosphate↗
PubMed1997

Oxidative activation of K-Cl cotransport by diamide in erythrocytes from humans with red cell disorders, and from several other mammalian species.

Red blood cells (RBCs) from different mammalian species were investigated for the presence of diamide-induced oxidative activation of K-Cl cotransport reported to be present in sheep but absent in human RBCs. K efflux was measured in RBCs from human with hemoglobin (Hb) A or S, glucose-phosphate dehydrogenase (G6PDH) and a cytoskeletal deficiency, and from rat, mouse and rabbit. RBCs were incubated with diamide (0-1.0 mm) in K-free Cl or NO3 media of variable osmolalities (200-450 mOsM). Cl-dependent K efflux or K-Cl cotransport (estimated as the difference between K efflux rate constants in Cl and NO3) was activated by diamide in a sigmoidal fashion. Relative maximum K-Cl cotransport followed the sequence: human HbA (1) < rabbit (1.8) < sheep (6.9) < human HbS (9.5) approximately rat (9.7). Relative diamide concentrations for half maximal activation of K-Cl cotransport followed the sequence: sheep (1.9) > human Hb A (1) > rabbit (0.75) > human HbS and rat (0.67). Cell swelling in 200 mOsM doubled K-Cl cotransport in diamide, both in human HbA and S cells but reduced that in rat RBCs. In contrast, cell shrinkage at 450 mOsM obliterated K-Cl cotransport in human HbA and S but not in rat RBCs. Human RBCs with G6PDH and a cytoskeleton deficiency behaved like HbA RBCs. In mouse RBCs, diamide-activated K-Cl cotransport was 30% higher in isotonic than in hypotonic medium. In human HbA and S, and in low or high K sheep RBCs fractionated by Percoll density gradient, diamide increased the activity of K-Cl cotransport, an effect inversely correlated with cell density. Analysis of pooled data reveals that K-Cl cotransport accounted for about 80% of all K flux in Cl. There was a statistically significant correlation between K-Cl cotransport and K efflux in Cl (P < 0. 00001) and in NO3 (P < 0.00001). In conclusion, a diamide-activated K-Cl cotransport was present in human RBCs and in all other mammalian RBCs tested, with a large inter-, and for human and sheep, intraspecies variability for its maximum activity.

Anemia, Sickle Cell↗
PubMed1996

A thermodynamic study of electroneutral K-Cl cotransport in pH- and volume-clamped low K sheep erythrocytes with normal and low internal magnesium.

Swelling-induced human erythrocyte K-Cl cotransport is membrane potential independent and capable of uphill transport. However, a complete thermodynamic analysis of basal and stimulated K-Cl cotransport, at constant cell volume, is missing. This study was performed in low K sheep red blood cells before and after reducing cellular free Mg into the nanomolar range with the divalent cation ionophore A23187 and a chelator, an intervention known to stimulate K-Cl cotransport. The anion exchange inhibitor 4,4'diisothiocyanato-2,2'disulfonic stilbene was used to clamp intracellular pH and Cl or NO3 concentrations. Cell volume was maintained constant as external and internal pH differed by more than two units. K-Cl cotransport was calculated from the K effluxes and Rb (as K congener) influxes measured in Cl and NO3, at constant internal K and external anions, and variable concentrations of extracellular Rb and internal anions, respectively. The external Rb concentration at which net K-Cl cotransport is zero was defined as flux reversal point which changed with internal pH and hence Cl. Plots of the ratio of external Rb concentrations corresponding to the flux reversal points and the internal K concentration versus the ratio of the internal and external Cl concentrations (i.e., the Donnan ratio of the transported ions) yielded slopes near unity for both control and low internal Mg cells. Thus, basal as well as low internal Mg-stimulated net K-Cl cotransport depends on the electrochemical potential gradient of KCl.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1996

Role of protein kinases in regulating sheep erythrocyte K-Cl cotransport.

K-Cl cotransport in sheep erythrocytes can be activated by treatment either with A-23187 and EDTA to reduce concentration of internal ionized Mg [Mg]i) to submicromolar levels, with staurosporine, a potent kinase inhibitor, or with N-ethylmaleimide (NEM). Activation by these maneuvers is prevented and reversed by genistein [inhibition constant (Ki) of 15 microM], which inhibits tyrosine kinases (TK). The related glycosidated compound genistin, which does not inhibit TK, does not inhibit transport, whereas another TK inhibitor, tyrphostin B46, inhibits both basal and stimulated transport (Ki of 28 microM). Cotransport activation by NEM is prevented and reversed by the phosphatase inhibitor, calyculin A, and activation by staurosporine occurs only if cells contain ATP. Increasing [Mg]i inhibits cotransport in the presence of calyculin A whether or not staurosporine is present as well. Our work suggests that genistein inhibits cotransport through a TK and that staurosporine and NEM activate cotransport, probably through inhibition of other kinases, causing stimulation through dephosphorylation of a protein (possibly the transporter itself) be a serine/threonine phosphatase. [Mg]i inhibits cotransport by activating a kinase (concentration for half-maximal activation of 10 microM) that phosphorylates this protein.

Adenosine Triphosphate↗
PubMed1996

Modulation of K-Cl cotransport in volume-clamped low-K sheep erythrocytes by pH, magnesium, and ATP.

Cellular pH, ionized Mg (Mgi2+), and MgATP concentration of red blood cells, concomitantly with cell volume, change transiently during circulation. The action of these three effectors on Cl-dependent K efflux was examined in low-K sheep red blood cells with constant cell volume. Activation of K-Cl efflux by Mgi2+ extraction required ATP, suggesting that phosphorylation of a putative component occurred before Mgi2+ extraction. Conversely, Mg and ATP were synergistic inhibitors of K-Cl cotransport, since maximal inhibition was observed only in cells containing both ATP and > 300 microM Mgi2+. Both findings suggest dual roles for Mg and ATP. At 300-600 microM Mgi2+, lowering the pH from approximately 7.4 to approximately 6.5 stimulated K-Cl efflux only in fed cells, suggesting that protons oppose or release the inhibition by Mgi2+ and ATP. A direct effect of both protons and Mgi2+ on the cotransporter is suggested by their inhibition of K-Cl efflux in ATP-depleted cells. These findings are discussed in light of the current phosphorylation/dephosphorylation hypothesis.

Adenosine Triphosphate↗
PubMed1995

Glutathione removal reveals kinases as common targets for K-Cl cotransport stimulation in sheep erythrocytes.

K-Cl cotransport is activated by swelling, lowering of cellular free Mg (Mgi), and thiol modification of erythrocytes. Direct actions by thiol reagents on the K-Cl cotransport complex were separated from indirect effects through nonoxidative changes in cellular glutathione (GSH). We used 1-chloro-2,4-dinitrobenzene (CDNB), which, conjugated to GSH, is extruded from the erythrocyte as a thioether. CDNB caused a small biphasic effect (inhibition and stimulation) on K-Cl cotransport and, at 1 mM, abolished its stimulation by N-ethylmaleimide (NEM), diazenedicarboxylic acid bis[N,N-dimethylamide], methyl methanethiosulfonate, and staurosporine, a kinase inhibitor, independent of the order of treatment. Hence, NEM and other activating-thiol reagents, and perhaps GSH removal itself, target unidentified kinases involved in activation of K-Cl cotransport. CDNB also abrogated K-Cl cotransport stimulation by Mgi depletion independent of the order of treatment, indicating inhibition at a second site nearer to the transporter. Furthermore, CDNB treatment elevated and rendered K-Cl cotransport insensitive to osmotic shrinkage, suggesting uncoupling from the regulator.

Adenosine Triphosphate↗
PubMed1995

Temperature-induced functional deocclusion of thiols inhibitory for sheep erythrocyte K-Cl cotransport.

In low-K sheep erythrocytes, K-Cl cotransport is activated by treatment with low concentrations of thiol reagents and by other interventions such as lowering of cellular free cytosolic Mg, hyposmotic cell swelling, the kinase inhibitor staurosporine, and hydroxylamine. High concentrations of N-ethylmaleimide or methylmethane thiolsulfonate reverse the activation through thiol groups and, as shown here, also the stimulation by non-thiol manipulations. The overriding inhibitory sites functionally associated with and different from those of the activating thiols (SHa) were further distinguished by temperature. Treatment with N-ethylmaleimide and its subsequent removal by dithiothreitol, both at 0 degrees C, prevented the inhibitory effect at 37 degrees C and thus the chemical modification of inhibitory thiols (SHi). Whereas stimulation through SHa closely followed the loss of glutathione, inhibition through SHi occurred only in glutathione-depleted cells. The reversal of K-Cl cotransport stimulation by all hitherto known interventions, which is strongest in metabolically depleted cells, suggests that the low temperature-protected SHi constitute crucial sites that, close to the transporter itself or at the cytoskeletal level, become functionally deoccluded upon temperature elevation.

Animals↗
PubMed1994

Hydroxyurea affects cell morphology, cation transport, and red blood cell adhesion in cultured vascular endothelial cells.

Hydroxyurea (HU) significantly increases fetal hemoglobin (Hb) production and concomitantly affects passive erythrocyte K transport and cell volume in patients homozygous for Hb S, thus decreasing disease severity. Red blood cells (RBCs) with Hb S display a greater adherence to vascular endothelial cells (VECs) than do Hb A cells, thus increasing the probability of vaso-occlusive crisis. The effect of HU on the structure and function of VECs is still unknown. In the present study, HU significantly changed, in a dose-dependent manner, the morphology and monovalent cation composition of cultured VECs after incubation in normal culture medium for up to 10 days in the absence and presence of 0.3 (therapeutic dose) and 3.0 (toxic dose) mmol/L HU. Treated cells showed significant morphologic changes such as an increase in apparent cell size and the formation of multinucleated giant cells. The protein content per dish decreased by 50% and 80% at 0.3 and 3.0 mmol/L HU, respectively, accompanied by an increase in cell Na (maximum, approximately 200%) and cell K (maximum, approximately 50%) contents at about days 4 to 6 and 8 to 10, respectively. In addition, HU decreased RBC adherence to VECs in experiments with 51Cr-loaded Hb A or Hb S RBCs. The HU-induced changes in VEC morphology, cation composition, and RBC adherence may be caused or accompanied by alterations in cell membrane permeability, transformation of endothelial cells, or decreased number/density of VEC adhesion molecules. Precise mechanisms of the HU effects warrant further investigation in light of the reported beneficial effects of HU in the treatment of sickle cell anemia.

Animals↗
PubMed1994

Quinine and quinidine inhibit and reveal heterogeneity of K-Cl cotransport in low K sheep erythrocytes.

Low K (LK) sheep red blood cells (SRBCs) serve as a model to study K-Cl cotransport which plays an important role in cellular dehydration in human erythrocytes homozygous for hemoglobin S. Cinchona bark derivatives, such as quinine (Q) and quinidine (QD), are effectively used in the treatment of malaria. In the present study, we investigated in LK SRBCs, the effect of various concentrations of Q and QD on Cl-dependent K efflux and Rb influx (K(Rb)-Cl flux), activated by either swelling in hyposmotic media, thiol alkylation with N-ethylmaleimide (NEM), or by cellular Mg (Mgi) removal through A23187 in the presence of external chelators. K efflux or Rb influx were determined in Cl and NO3 medium and K(Rb)-Cl flux was defined as the Cl-dependent (Cl minus NO3) component. K(Rb)-Cl flux stimulated by all three interventions was inhibited by both Q and QD in a dose-dependent manner. Maximum inhibition of K(Rb)-Cl flux occurred at Q and QD concentrations > or = 1 mM. The inhibitory effect of Q was manifested in Cl, but not in NO3, whereas QD reduced K and Rb fluxes both in Cl and NO3 media. The mean 50% inhibitory concentration (IC50) of Q and QD to inhibit K(Rb)-Cl flux varied between 0.23 and 2.24 mM. From determinations of the percentages of inhibition of the different components of K and Rb fluxes, we found that SRBCs possess a Cl-dependent QD-sensitive and a Cl-dependent QD-insensitive K efflux and Rb influx. These two components vary in magnitude depending on the manipulation and directional flux, but in average they are about 50% of the total Cl-dependent flux. This study raises the possibility that, in SRBCs, the Cl-dependent K(Rb) fluxes are heterogeneous.

Animals↗
PubMed1994

K-Cl cotransport, pH, and role of Mg in volume-clamped low-K sheep erythrocytes: three equilibrium states.

Ouabain-resistant K efflux and Rb influx in Cl and NO3 media were studied in volume-clamped low-K (LK) sheep red blood cells (SRBC) with normal and experimentally reduced cytoplasmic Mg (Mgi) levels as function of pH and at 37 degrees C. Sucrose was added to solutions with constant ionic strength and variable pH to maintain normal cell volume. Cl-dependent ouabain-resistant K(Rb) fluxes (K-Cl cotransport) at unity relative cell volume exhibited a maximum at pH approximately 7 in normal-Mgi LK cells consistent with the apparent acid pH activation reported for human erythrocytes. However, in LK SRBC with Mgi lowered by A-23187 and an external Mg chelator, K(Rb)-Cl cotransport was reversibly activated as the pH was raised from 6.5 to 9. The alkaline pH effect on Cl-dependent Rb influx in low-Mgi LK SRBC was due to a 10-fold rise in the maximum velocity values without a major change in the Km values. The pH dependence of the experimental flux reversal point, i.e., the extracellular Rb concentration at which no net K-Cl cotransport occurs, approximately paralleled that of the flux reversal point predicted from the ratio of the ion products, in both control and low-Mgi LK cells, albeit with a small displacement to higher extracellular Rb concentration at all pH values. The kinetic data can be explained by a general minimum three-state equilibrium in which deprotonation recruits transporters from a resting R state into the active A state modified by Mgi to an inactive I state.(ABSTRACT TRUNCATED AT 250 WORDS)

Acid-Base Equilibrium↗
PubMed1992

Incorporation of 3H-N-ethylmaleimide into sheep red cell membrane thiol groups following protection by diamide-induced oxidation.

The thiol oxidant diazene dicarboxylic acid bis [N,N-dimethylamide] (diamide) is known to reversibly activate K-Cl cotransport in sheep red blood cells. Although the detailed mechanism of activation is unknown, functional thiols at the membrane or at the cytoplasmic level are recognized as important. To search for membrane bound thiols involved in the regulation of K-Cl cotransport, sheep red cells were first exposed to diamide at concentrations activating K-Cl cotransport, and then to the alkylating agent N-ethylmaleimide (NEM) in order to block non-oxidized thiols. White ghosts, prepared by osmotic lysis from these cells, were again treated with NEM followed by reduction of the diamide-induced dithiols with dithiothreitol (DTT) concentrations known to reverse the diamide-induced K-Cl flux. Maximum 3H-NEM incorporation into the DTT-reduced thiols occurred at 50 microM DTT. Saturation labelling by 3H-NEM of about 2 x 10(4) diamide-protected thiols/cell occurred at 25 microM NEM. Diamide protected about 0.1% of all membrane thiols chemically determined earlier. Membranes from high K (HK) and low K (LK) sheep red cells did not differ significantly in the number of diamide-protected thiols, and polyacrylamide gels revealed a similar protein distribution of 3H-NEM-labelled thiols. Since diamide is known to stimulate K-Cl flux in LK cells ten times more than in HK cells this finding is consistent with the hypothesis of a cytoplasmic control effecting different K-Cl flux activities in the membranes of the two cation genotypic red blood cells.

Animals↗
PubMed1992

Kinetics of DIDS inhibition of swelling-activated K-Cl cotransport in low K sheep erythrocytes.

The inhibitory effect of various stilbene disulfonates was examined on the swelling-activated Cl-dependent K transport (K-Cl cotransport) in low K sheep erythrocytes. Both diisothiocyanatostilbenes H2DIDS and DIDS were found to be potent inhibitors. The DIDS concentration yielding 50% inhibition (IC50) of KCl cotransport was 60 microM in the absence of external K and 3 microM at physiological K concentration. Other stilbene derivatives, such as SITS (4-acetamido-4' isothiocyanatostilbene-2,2'-disulfonic acid), were only effective in the presence of external K, whereas DNDS (4,4'-dinitrostilbene-2,2'-disulfonic acid) and ISA (4-sulfophenyl isothiocyanate) had only slight effects at a concentration of 1 mM. The augmenting effect of external K is due to a second K site, distinguishable from the K transport site by its much higher affinity. No inhibition occurred in the absence of external Cl, whether or not external Rb(K) was present. Additionally, DIDS inhibited K-Cl cotransport activated by thiol alkylation with N-ethylmaleimide (NEM) as well as by Mg depletion in the presence of A23187 and a chelator. We conclude that allosteric sites affect the stilbene binding. When these sites are saturated, changes in external K or Cl concentration do not affect the affinity for DIDS (noncompetitive inhibition).

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1992

Erythrocyte K-Cl cotransport: properties and regulation.

Erythrocytes possess a Cl-dependent, Na-independent K transport system cotransporting K and Cl in a 1:1 stoichiometry that is membrane potential independent. This K-Cl cotransporter is stimulated by cell swelling, acidification, Mg depletion, and thiol modification. Cell shrinkage, elevation of cellular divalent ions, thiol alkylation, phosphatase inhibitors, and derivatives of certain loop diuretics and stilbenes are inhibitory. Thus regulation of K-Cl cotransport at the membrane and cytoplasmic levels is highly complex. Basal K-Cl cotransport decreases with cellular maturation, whereas its modes of stimulation and inhibition are variable between species. The physiological inactivation appears to be prevented in low-K animal erythrocytes. In certain human hemoglobinopathies, K-Cl cotransport may be the cause of cellular dehydration and volume decrease. K-Cl cotransport occurs also in nonerythroid cells, such as in epithelial and liver cells of other species. At the threshold of molecular characterization, this comprehensive review places our present understanding of the mechanisms modulating K-Cl cotransport physiologically and pathophysiologically into kinetic and thermodynamic perspectives.

Animals↗
PubMed1991

Relationship between total magnesium concentration and free intracellular magnesium in sheep red blood cells.

The cellular free magnesium concentration of ionophore A23187 permeabilized high potassium sheep erythrocytes was measured by 31P nuclear magnetic resonance spectroscopy, and the total cellular magnesium concentration was determined by atomic absorption spectroscopy. The free versus total cellular magnesium concentrations yield a linear relationship on a log-log scale in the concentration range from 0.3 to 1.92 mmol Mg/liter cells. Thus, free intracellular magnesium concentrations can be calculated from atomic absorption data. The method permits the estimation of physiologically or experimentally induced variations of intracellular free magnesium concentrations between 7 and 405 microM magnesium in cell water. This range encompasses the free magnesium concentration of 335 +/- 60 microM in cell water determined for untreated erythrocytes.

Adenosine Triphosphate↗