Gain Of Na Research Articles

Transcriptomic changes in C2C12 myotubes triggered by electrical stimulation: Role of Ca2+i-mediated and Ca2+i-independent signaling and elevated [Na+]i/[K+]i ratio

Elevation of Ca2+i and AMP-activated protein kinase (AMPK) are considered as major signals triggering transcriptomic changes in exercising skeletal muscle. Electrical pulse stimulation (EPS) of cultured myotubes is widely employed as an in vitro model of muscle contraction. This study examines the impact of Ca2+i-mediated and Ca2+i-independent signaling in transcriptomic changes in EPS-treated C2C12 myotubes. Electrical pulse stimulation (40 V, 1 Hz, 10 ms, 2 h) resulted in [Ca2+]i oscillations, gain of Na+i, loss of K+i, and differential expression of 3215 transcripts. Additions of 10 μM nicardipine abolished [Ca2+]i oscillations but did not affect elevation of the [Na+]i/[K+]i ratio seen in EPS-treated myotubes. Differential expression of 1018 transcripts was preserved in the presence of nicardipine, indicating a Ca2+i-independent mechanism of excitation–transcription coupling. Among nicardipine-resistant transcripts, we noted 113 transcripts whose expression was also affected by partial Na+,K+-ATPase inhibition with 30 μM ouabain providing the same elevation of the [Na+]i/[K+]i ratio as in EPS-treated cells. Electrical pulse stimulation increased phosphorylation of CREB, ATF-1, Akt, ERK, and p38 MAPK without any impact on phosphorylation of acetyl-CoA carboxylase and Unc-51 like autophagy activating kinase-1, i.e. downstream markers of AMPK activation. Unlike CREB, ATF-1, and MAPKs, an increment in Akt phosphorylation was abolished by nicardipine. Thus, our results show that Ca2+i-independent signaling plays a key role in altered expression of 30% of studied genes in EPS-treated myotubes. This signaling pathway is at least partially triggered by dissipation of transmembrane gradients of monovalent cations.

New evidence for two sharp replacement fronts during albitization of granitoids from northern Aravalli orogen, northwest India

We present new evidence of infiltration metasomatism in granitoids that were albitized in a process that produced two sharp replacement fronts, both of which are clearly visible in the field. The two fronts advanced through the original granite simultaneously, but at different rates. Here we focus mainly on the Ajitgarh intrusive in the northern Aravalli orogen of northwest India. This intrusion shows geographically well-defined metasomatic zones on the outcrop scale as well as a large volume of original ferroan granite, both of which were poorly preserved in most of the previously studied Khetri granites. Stage I metasomatism transformed the grey original granite to pink microcline–albite granite, and stage II converted the microcline–albite granite to white albite granite. Both these reaction fronts are sharp and are easily recognized in the field by their different colours. The mineralogical and chemical changes during the first stage are expressed by transformation of original oligoclase to albite, biotite (annite-rich) and hastingsite (amphibole) to hastingsite with low XFe values, dehydration, gain in Na, and losses in Fe and Rb. The second stage of metasomatism caused almost complete conversion of microcline to albite and complete or nearly complete disappearance of amphibole. Chemically, these changes are manifested by substantial gain in Na and extreme losses in K, Rb, Ba, Ca, Sr, Fe, and Mg. Depending on the modal abundances of amphibole, stage II albitized rocks are depleted in light rare earth elements or heavy rare earth elements or both, signifying that rare earth elements are principally hosted by mafic phases. The disparity in whole-rock δ18O values during both stages of albitization is related to the variations in modal amounts of Si-bearing phases. The replacement microstructures are in accord with the fluid-mediated phase transformations by a coupled dissolution–precipitation mechanism. The albitizing event took place at low temperatures of 350–400 °C and the fluid was metamorphic in nature.

Effects of β2-agonists on force during and following anoxia in rat extensor digitorum longus muscle

Electrical stimulation of isolated muscles may lead to membrane depolarization, gain of Na(+), loss of K(+) and fatigue. These effects can be counteracted with β(2)-agonists possibly via activation of the Na(+)-K(+) pumps. Anoxia induces loss of force; however, it is not known whether β(2)-agonists affect force and ion homeostasis in anoxic muscles. In the present study isolated rat extensor digitorum longus (EDL) muscles exposed to anoxia showed a considerable loss of force, which was markedly reduced by the β(2)-agonists salbutamol (10(-6) M) and terbutaline (10(-6) M). Intermittent stimulation (15-30 min) clearly increased loss of force during anoxia and reduced force recovery during reoxygenation. The β(2)-agonists salbutamol (10(-7)-10(-5) M) and salmeterol (10(-6) M) improved force development during anoxia (25%) and force recovery during reoxygenation (55-262%). The effects of salbutamol on force recovery were prevented by blocking the Na(+)-K(+) pumps with ouabain or by blocking glycolysis with 2-deoxyglucose. Dibutyryl cAMP (1 mM) or theophylline (1 mM) also improved force recovery remarkably. In anoxic muscles, salbutamol decreased intracellular Na(+) and increased (86)Rb uptake and K(+) content, indicating stimulation of the Na(+)-K(+) pumps. In fatigued muscles salbutamol induced recovery of excitability. Thus β(2)-agonists reduce the anoxia-induced loss of force, leading to partial force recovery. These data strongly suggest that this effect is mediated by cAMP stimulation of the Na(+)-K(+) pumps and that it is not related to recovery of energy status (PCr, ATP, lactate).

Protective Effect of Cimetidine on Tannic Acid-induced Gastric Damage in Rats

The effect of cimetidine on the acidified tannic acid-induced back diffusion of gastric acid and mucosal damage was investigated in the vagotomized rat. After intragastric irrigation for 1 to 3 h, tannic acid (20-500 mg kg-1) produced a dose-related increase in gastric volume and the loss of luminal H+. The change of mucosal permeability to the electrolyte, either the loss of H+ or the gain of Na+, K+, and Ca2+, induced by tannic acid was significantly diminished by the combination with intragastric cimetidine. However, intravenous injection of cimetidine did not protect this damage and back diffusion of acid neutralized intragastric cimetidine did not reduce the back diffusion of acid and Na+, K+, and Ca2+ output provoked by acid solution. Thus, the neutralizing action of cimetidine seems responsible.

In isolated skeletal muscle, excitation may increase extracellular K+10-fold; how can contractility be maintained?

During excitation, isolated rat muscles undergo a net loss of cellular K(+), which cannot be cleared via capillaries and can only be cleared to a small extent via diffusion into the surrounding buffer. Therefore, K(+) accumulates in the extracellular space and can be quantified using flame photometry and compared with contractile performance in extensor digitorum longus (EDL) and soleus muscles. During electrical stimulation, extracellular potassium concentration ([K(+)](o)) shows a rapid early rise, followed by a slower increase, reflecting progressive loss of excitability. Thus, after 30 s of 60 Hz stimulation in EDL, where [K(+)](o) reaches 50 mm, increasing the pulse duration from 0.2 to 1.0 ms augments force by 172%. Excitation-induced cellular loss of K(+) coincides with an equivalent gain of Na(+), in keeping with the one-for-one exchange of Na(+) and K(+). This Na(+)-K(+) exchange is completely reversible and is not accompanied by changes in the (14)C-sucrose space or release of intracellular enzyme activity, indicating that it is not due to unspecific cellular leakage. In EDL, 60 s of 20 Hz stimulation induced force elevation and increased [K(+)](o) to 43-47 mm. The force elevation was suppressed by ouabain (10(-5)m), indicating that the Na(+)-K(+) pump contributes to maintenance of excitability. After 15 s of 60 Hz stimulation, resting net re-extrusion of Na(+) was 7.3-fold faster than basal Na(+) efflux. Bumetanide, which blocks Na(+)-K(+)-2Cl(-) cotransport, caused no change in force and K(+) contents, excitation-induced loss of K(+) or postexcitatory reaccumulation of K(+). Omission of Cl(-) increased the rate of force decline 14-fold. In conclusion, during repeated excitation, isolated rat muscles undergo a much greater increase in [K(+)](o) than previously reported, sufficient to explain loss of force. This K(+) is not cleared via Na(+)-K(+)-2Cl(-) cotransport, but rather via Na(+)-K(+) pumps and by processes depending on Cl(-) exchange. These mechanisms are essential for the maintenance of force during intense contractions in vivo, where the clearance of K(+) via the capillaries may be suppressed.

Ion channel inhibitors block caspase activation by mechanisms other than restoring intracellular potassium concentration

Ion fluxes at the plasma membrane have an important role in early stages of apoptosis. Accordingly, plasma membrane depolarization and gain of Na+ and loss of K+ are initial events in apoptosis. We have studied the effect of staurosporine (STS), a well-established apoptosis inducer, on the membrane potential of HeLa cells to determine the nature of STS-activated ion conductances and their role in the activation of different caspases. We observed that STS can activate tetraethylammonium (TEA+) and 4-aminopyridine-sensitive K+ channels and flufenamic-sensitive cation channels as an early response. The combination of these ion channel inhibitors significantly reduced cytochrome c (cyt c) release and activation of caspase-9, -3 and -8. STS also induced a large reduction in the intracellular [K+] that was not blocked by the ion channel inhibitors. Our data suggest that reduction in the [K+]i is necessary but not sufficient and that ion channel inhibitors block activation of caspase-3 by two different mechanisms: the inhibitors of K+ channels by reducing cyt c release while flufenamic acid by a different, unrelated mechanism that does not involve cation channels at the plasma membrane. Our data also imply that these ion channels activated by STS are not responsible for the reduction in the [K+]i associated with apoptosis.

Functional Roles of a Novel Structural Element Involving the Na+−π Interaction Present in the Catalytic Site of T1 Lipase Revealed by Molecular Dynamics Simulations

The interaction between a cation and an aromatic ring, i.e., the cation-π interaction, is one of the strongest noncovalent forces. Metal cations such as Na+ and K+ can also participate in the cation-π interactions, and are known to yield significant stabilization energy. However, in biological systems, few structures containing metal-π coordination have been determined, preventing understanding of its biological roles. Recently, we have determined the crystal structure of a thermoalkalophilic lipase where a Na+ is coordinated to a phenylalanine (Phe) in its catalytic site. To elucidate the functional roles of the Na+−Phe complex, we performed molecular dynamics (MD) simulations of the system. Note that the current force fields cannot correctly estimate the metal-π interaction energy, requiring quantum mechanical calculations. However, their huge computational costs prohibit long-time MD simulations. Accordingly, we developed a novel scheme to calculate the interaction energy with an accuracy comparable to that of advanced ab initio calculations at the CCSD(T) levels, and with computational costs comparable to those of force field calculations.A comparison of the MD simulations in the presence/absence of the accurate description revealed that a significantly large enthalpy gain in Na+−Phe substantially stabilizes the catalytic site. Thereby, the cation−π interaction in the lipase establishes a remarkably stable core structure by combining a hydrophobic aromatic ring and hydrophilic residues, of which the latter form the catalytic triad, thus contributing to large structural changes from the complex with ligands to the free form of the lipase. Thus, we have elucidated the detailed functional roles of Na+−π complex with use of our presented scheme, which is currently the only way to perform long-time MD simulations with reasonable computational costs.

Functional Roles of a Structural Element Involving Na+-π Interactions in the Catalytic Site of T1 Lipase Revealed by Molecular Dynamics Simulations

Interactions between metal ions and pi systems (metal-pi interactions) are known to confer significant stabilization energy. However, in biological systems, few structures with metal-pi coordination have been determined; thus, its roles must still be elucidated. The cation-pi interactions are not correctly described by current molecular mechanics even when using a polarizable force field, and thus they require quantum mechanical calculations for accurate estimation. However, the huge computational costs of the latter methodologies prohibit long-time molecular dynamics (MD) simulations. Accordingly, we developed a novel scheme to obtain an effective potential for calculating the interaction energy with an accuracy comparable to that of advanced ab initio calculations at the CCSD(T) levels, and with computational costs comparable to those of conventional MM calculations. Then, to elucidate the functional roles of the Na(+)-phenylalanine (Phe) complex in the catalytic site of T1 lipase, we performed MD simulations in the presence/absence of the accurate Na(+)-pi interaction energy. A comparison of these MD simulations revealed that a significantly large enthalpy gain in Na(+)-Phe16 substantially stabilizes the catalytic site, whereas a water molecule could not be substituted for Na(+) for sufficient stabilization energy. Thus, the cation-pi interaction in the lipase establishes a remarkably stable core structure by combining a hydrophobic aromatic ring and hydrophilic residues, of which the latter form the catalytic triad, thereby contributing to large structural changes from the complex with ligands to the free form of the lipase. This is the first report to elucidate the detailed functional mechanisms of Na(+)-pi interactions.

Ventricular performance and Na+-K+ ATPase activity are reduced early and late after myocardial infarction in rats

Myocardial infarction leads to compensatory ventricular remodeling. Disturbances in myocardial contractility depend on the active transport of Ca2+ and Na+, which are regulated by Na+-K+ ATPase. Inappropriate regulation of Na+-K+ ATPase activity leads to excessive loss of K+ and gain of Na+ by the cell. We determined the participation of Na+-K+ ATPase in ventricular performance early and late after myocardial infarction. Wistar rats (8-10 per group) underwent left coronary artery ligation (infarcted, Inf) or sham-operation (Sham). Ventricular performance was measured at 3 and 30 days after surgery using the Langendorff technique. Left ventricular systolic pressure was obtained under different ventricular diastolic pressures and increased extracellular Ca2+ concentrations (Ca2+e) and after low and high ouabain concentrations. The baseline coronary perfusion pressure increased 3 days after myocardial infarction and normalized by 30 days (Sham 3 = 88 +/- 6; Inf 3 = 130 +/- 9; Inf 30 = 92 +/- 7 mmHg; P < 0.05). The inotropic response to Ca2+e and ouabain was reduced at 3 and 30 days after myocardial infarction (Ca2+ = 1.25 mM; Sham 3 = 70 +/- 3; Inf 3 = 45 +/- 2; Inf 30 = 29 +/- 3 mmHg; P < 0.05), while the Frank-Starling mechanism was preserved. At 3 and 30 days after myocardial infarction, ventricular Na+-K+ ATPase activity and contractility were reduced. This Na+-K+ ATPase hypoactivity may modify the Na+, K+ and Ca2+ transport across the sarcolemma resulting in ventricular dysfunction.

Na+]i‐induced c‐Fos expression is not mediated by activation of the 5′‐promoter containing known transcriptional elements

In vascular smooth muscle cells and several other cell types, inhibition of Na(+)/K(+)-ATPase leads to the expression of early response genes, including c-Fos. We designed this study to examine whether or not a putative Na(+) (i)/K(+) (i)-sensitive element is located within the c-Fos 5'-UTR from - 650 to + 103 containing all known response elements activated by 'classic' stimuli, such as growth factors and Ca(2+) (i)-raising compounds. In HeLa cells, the highest increment of c-Fos mRNA content was noted after 6 h of Na(+)/K(+)-ATPase inhibition with ouabain that was abolished by actinomycin D, an inhibitor of RNA synthesis. c-Fos protein accumulation in ouabain-treated cells correlated with a gain of Na(+) (i) and loss of K(+) (i). Augmented c-Fos expression was also observed under inhibition of Na(+)/K(+)-ATPase in K(+)-free medium and in the presence of the Na(+) ionophore monensin. The effect of ouabain on c-Fos expression was sharply attenuated under dissipation of the transmembrane Na(+) gradient, but was preserved in the presence of Ca(2+) chelators and the extracellular regulated kinase inhibitor PD98059, thus indicating an Na(+) (i)-mediated, Ca(2+) (i)- and extracellular regulated kinase-independent mechanism of gene expression. In contrast to massive c-Fos expression, we failed to detect any effect of ouabain on accumulation of luciferase driven by the c-Fos 5'-UTR. Negative results were also obtained in ouabain-treated vascular smooth muscle cells and C11 Madin-Darby canine kidney cells possessing augmented c-Fos expression. Our results reveal that Na(+) (i)-induced c-Fos expression is not mediated by the 5'-UTR containing transcriptional elements activated by growth factors and other 'classic stimuli'.

Excitation-induced cell damage and β2-adrenoceptor agonist stimulated force recovery in rat skeletal muscle

Intensive exercise leads to a loss of force, which may be long lasting and associated with muscle cell damage. To simulate this impairment and to develop means of compensating the loss of force, extensor digitorum longus muscles from 4-wk-old rats were fatigued using intermittent 40-Hz stimulation (10 s on, 30 s off). After stimulation, force recovery, cell membrane leakage, and membrane potential were followed for 240 min. The 30-60 min of stimulation reduced tetanic force to approximately 10% of the prefatigue level, followed by a spontaneous recovery to approximately 20% in 120-240 min. Loss of force was associated with a decrease in K+ content, gain of Na+ and Ca2+ content, leakage of the intracellular enzyme lactic acid dehydrogenase (10-fold increase), and depolarization (13 mV). Stimulation of the Na+-K+ pump with either the beta2-adrenoceptor agonist salbutamol, epinephrine, rat calcitonin gene-related peptide (rCGRP), or dibutyryl cAMP improved force recovery by 40-90%. The beta-blocker propranolol abolished the effect of epinephrine on force recovery but not that of CGRP. Both spontaneous and salbutamol-induced force recovery were prevented by ouabain. The salbutamol-induced force recovery was associated with repolarization of the membrane potential (12 mV) to the level measured in unfatigued muscles. In conclusion, in muscles exposed to fatiguing stimulation leading to a considerable loss of force, cell leakage, and depolarization, stimulation of the Na+-K+ pump induces repolarization and improves force recovery, possibly due to the electrogenic action of the Na+-K+ pump. This mechanism may be important for the restoration of muscle function after intense exercise.

Regional-scale fluid flow and element mobility in Barrow’s metamorphic zones, Stonehaven, Scotland

The geochemistry of metamorphic quartz vein formation in Barrow’s index mineral zones north of Stonehaven, Scotland, was investigated in order to assess regional fluid flow and mass transfer. Metamorphic grade in the Dalradian metasediments increases to the north–northwest away from the Highland Boundary Fault (HBF) and associated ophiolitic rocks of the Highland Border Complex (HBC), passing through the Chlorite (Chl), Biotite (Bt), Garnet (Grt), Chloritoid (Cld), and Staurolite (St) zones. Syn-metamorphic fluid infiltration at 462±8.8 Ma (Breeding et al. in Am Mineral 89:1067, 2004) produced considerable quartz veining. Vein abundance varies from about 5 to 15 volume percent of the outcrops; veins tend to be more abundant in metapelitic layers than in metapsammitic ones. Metamorphic veins are surrounded by centimeter- to decimeter-wide zones of chemical and mineralogical alteration (selvages). Porphyroblasts, particularly Bt, Grt, Cld, and St, are typically larger in selvages than in wallrocks distal to veins. The altered selvages underwent fluid-driven addition of Na, Ca, and Sr, and loss of K, Rb, and Ba. Alteration is most intense within ∼750 m of the HBF, but is still very significant at the northern end of the field area some 2 km away. Mg/FeT (FeT=total iron) was either unchanged or increased due to alteration. Silica was added at some Chl and Bt zone localities near the HBF. Pb mass transfer was variable although Pb was added at a number of locations. Rare Earth elements (REE) were generally immobile, but light REE and possibly heavy REE were lost at one field site. The gain of Na and Ca and loss of K promoted the growth of plagioclase at the expense of micas (particularly muscovite) in selvages and wallrock inclusions throughout the field area and, probably, some calcite and/or dolomite growth directly adjacent to the HBF. The Ca gains were also critical for epidote production in the Bt zone. Gains of Ca and increases in Mg/FeT helped to stabilize Grt at the expense of Cld and St in some selvages. Hornblende and cummingtonite were discovered in strongly altered metapelitic rocks at one Cld zone locality. The metasomatism puts important constraints on the processes of mass transfer and suggests two models for regional fluid flow. In the first model, fluid flow in a direction of increasing temperature downward along the HBF added Na and Ca, and removed K from the Dalradian. In the second model, fluid flow upward from the HBC transported Na and Ca into the overlying Dalradian and, at the same time, stripped out K. The latter model is favored because it can most readily account for silica addition near the HBF, but neither model can be ruled out at present. In either case, the veins represent fractures that transmitted very large time-integrated fluid fluxes of at least ∼104 m3 (fluid)/m2 (rock). Consequently, the veins were conduits for regional fluid flow that caused considerable open-system chemical and mineralogical alteration during metamorphism.

Role of Na+,K+ pumps in restoring contractility following loss of cell membrane integrity in rat skeletal muscle

In skeletal muscles, electrical shocks may elicit acute loss of force, possibly related to increased plasma membrane permeability, induced by electroporation (EP). We explore the role of the Na(+),K(+) pumps in force recovery after EP. Isolated rat soleus or extensor digitorum longus (EDL) muscles were exposed to EP paradigms in the range 100-800 V cm(-1), and changes in tetanic force, Na(+),K(+) contents, membrane potential, (14)C-sucrose space and the release of the intracellular enzyme lactic acid dehydrogenase (LDH) were characterized. The effects of Na(+),K(+) pump stimulation or inhibition were followed. Electroporation caused voltage-dependent loss of force, followed by varying rates and degrees of recovery. EP induced a reversible loss of K(+) and gain of Na(+), which was not suppressed by tetrodotoxin, but associated with increased (14)C-sucrose space and release of LDH. In soleus, EP at 500 V cm(-1) induced complete loss of force, followed by a spontaneous, partial recovery. Stimulation of active Na(+),K(+) transport by adrenaline, the beta(2)-agonist salbutamol, calcitonin gene-related peptide (CGRP) and dibutyryl cyclic AMP increased initial rate of force recovery by 183-433% and steady-state force level by 104-143%. These effects were blocked by ouabain (10(-3) m), which also completely suppressed spontaneous force recovery. EP caused rapid and marked depolarization, followed by a repolarization, which was accelerated by salbutamol. Also in EDL, EP caused complete loss of force, followed by a spontaneous partial recovery, which was markedly stimulated by salbutamol. Electroporation induces reversible depolarization, partial rundown of Na(+),K(+) gradients, cell membrane leakage and loss of force. This may explain the paralysis elicited by electrical shocks. Na(+),K(+) pump stimulation promotes restoration of contractility, possibly via its electrogenic action. The major new information is that the Na(+),K(+) pumps are sufficient to compensate a simple mechanical leakage. This may be important for force recovery in leaky muscle fibres.

The sodium pump keeps us going.

This invited lecture reviews recent evidence that, in skeletal muscle, excitability and contractility depend on the transmembrane distribution of Na(+) and K(+) and the membrane potential, which in turn are determined by the operation of the Na(+)-K(+) pump. Action potentials are elicited by passive fluxes of Na(+) and K(+). Because of their size and sudden onset, these transport events constitute the major challenge for the Na(+)-K(+) pumps. When the Na(+)-K(+) pumps cannot readily restore the Na(+)-K(+) gradients, working muscle cells often undergo net loss of K(+) and gain of Na(+). This leads to loss of excitability and force, in particular, in muscles where excitation-induced passive Na(+)-K(+) fluxes are large. Thus, excitability depends on the leak/pump ratio for Na(+) and K(+). When this ratio is increased by inhibition or downregulation of the Na(+)-K(+) pumps, the force decline seen during continued stimulation is accelerated. This effect is highly significant already within the first seconds of electrical stimulation. Fortunately, electrical stimulation also increases Na(+)-K(+) pumping rate within seconds. Thus, maximum increase (20-fold above the resting level) may be reached in 10 seconds, with utilization of all available Na(+)-K(+) pumps. In muscles, where excitability was inhibited by exposure to high [K(+)](o) (10-12.5 mM), activation of the Na(+)-K(+) pumps by hormones or electrical stimulation restored excitability and contractile force. In working muscles, the Na(+)-K(+) pumps, because of rapid activation of their large transport capacity, play a dynamic regulatory role in the second-to-second ongoing restoration and maintenance of excitability and force. The Na(+)-K(+) pumps become a limiting factor for contractile endurance, in particular, if their capacity is reduced by inactivity or disease.

Bisphenolic compounds that enhance cell cation transport are found in commercial phenol red.

We have isolated two bisphenolic compounds (4 and 5) that have a marked effect on K+ and Na+ concentrations in human cells from commercial preparations of the pH indicator dye phenol red (phenolsulfonphthalein). We used a bioassay to identify active chromatographic fractions from the lipophilic impurities present in phenol red, and we determined the structure of two active components (4 and 5) by 1H and 13C NMR and mass spectrometry. When added to human fibroblasts in serum-free medium, the bisphenol fluorene derivative 9,9-bis(4'-hydroxyphenyl)-3-hydroxyfluorene (5) produced a rapid loss of K+ and a gain of Na+, at low concentrations, with an EC50 between 30 and 60 ng/mL (80-160 nM). The 2- and 4-hydroxy isomers of the fluorene 5 (i.e., compounds 6 and 7), prepared by synthesis, had similar activity, although compound 6 was somewhat less potent. The bisphenol xanthene derivative 9,9-bis(4'-hydroxyphenyl)xanthene (4) elicited a similar biological response but was less potent than 5-7; it also had a strong effect on cell adhesion, causing release of cells from the plastic substrate at concentrations as low as 2-5 microg/mL (5.5-14 microM). The structures of xanthene (4) and fluorene (5) bisphenols have been confirmed by synthesis from xanthone and hydroxyfluorenone, respectively, by Friedel-Crafts alkylation with phenol. In the latter case, the desired 3-hydroxyfluorene isomer was formed in situ by rearrangement of the 1-hydroxy isomer.

Effect of non-lytic concentrations of Brij series detergents on the metabolism-independent ion permeability properties of human erythrocytes

Subcritical micellar concentrations (sub-CMC) of Brij-series detergents alter ion movements between human erythrocytes and their environment when metabolism has been slowed down by incubation at zero degrees centigrade. The effect of nonhemolytic concentrations of detergents on the erythrocyte K+ and Na+ movements is described. Results indicate a significant difference in monovalent cation movements, depending on the number of hydrophilic polyoxyethylene units (n). There is an increasing loss of K+ and gain of Na+ as n increases from 4 to 20. Where n > or = 21, ion movements are not significantly different from those found in erythrocytes not exposed to detergents. The carbon chain length of the detergent fatty acid residue (10-18 carbons) appears to be relatively unimportant, but detergents with unsaturated (oleic acid) hydrophobic regions potentiate K+ release and Na+ uptake when compared to the corresponding saturated fatty acid (stearic acid). The erythrocyte stabilizing effect of detergents against hypo-osmotic shock correlates well with the increase of monovalent ion traffic and the mobility of membrane lipids revealed by fluorescence anisotropy measurements.

Effects of voltage clamping on epithelial cell composition in toad urinary bladder studied with x-ray microanalysis.

Toad urinary bladder epithelial cells were incubated in Na Ringer's with the serosal surface of the epithelium clamped at either +50 mV, 0 mV (short-circuited) or -50 mV with respect to the mucosal surface. Following incubation, portions of tissue were coated with an external albumin standard and rapidly frozen. Cryosections were freeze-dried and cell composition determined by x-ray microanalysis. Cell water and ion contents were unaffected when tissues were short-circuited rather than clamped close to their open-circuit potential difference (+50 mV). Incubation with vasopressin at +50 mV, and under short-circuit conditions, caused Na uptake without cell swelling or gain in Cl. Clamping at -50 mV resulted in uptake of water and ions, with considerable variation from cell to cell. These variations in cell composition were exacerbated by vasopressin. The greater the increase in water content, the greater the rise in cell Cl. However, there was no consistent pattern to the associated changes in cation contents. Most cells gained some Na. In some cells, this gain was accompanied by an increase in K. In others, the gain of Na was predominant and cell K content actually fell. At -50 mV with ouabain, many of the cells also gained water. As was found in our earlier study with ouabain under short circuit conditions (Bowler et al., 1991), there was considerable variation in the extent of the Na gain and K loss; some cells were largely depleted of K while in others the K content remained relatively normal. These results indicate differences between granular cells in the availabilities in the plasma membranes of ion pathways, either as a consequence of differences in the numbers of such pathways or in their control.

Calcium-activated sodium and chloride fluxes modulate platelet volume: role of Ca2+ stores

An increase in cytosolic ionized Ca2+ concentration ([Ca2+]i) initiates volume changes in various types of cells. In response to increases in [Ca2+]i most cell types contract by efflux of K+ and Cl-, whereas platelets expand in response to rises in [Ca2+]i. This study examined the importance of the source of Ca2+, the flux of ions responsible for the volume change, and the role of Ca(2+)-dependent protein kinases in regulating these ionic fluxes. The baseline platelet volume was independent of extracellular Ca2+ but when stimulated by the Ca2+ ionophore A-23187 (50 nM) the volume increased in both the presence and absence of extracellular Ca2+ (1.18 +/- 0.08 vs. 0.83 +/- 0.06 fl, respectively). The increased volume was caused by the gain of Na+ and Cl-. Na+ entered through both conductive and nonconductive (Na+/H+ exchange) pathways, whereas the influx of Cl- was conductive and inhibited by the Cl- channel blocker 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. The Ca(2+)-induced volume change was blocked by both calmodulin and protein kinase inhibitors. Thus the activation of Ca(2+)-dependent protein kinases promotes platelet swelling by stimulating Na+ and Cl- influx.

Changes in K+, Na+ and calcium contents during in vivo stimulation of rat skeletal muscle.

The effects of in vivo stimulation via the sciatic nerve on Na+, K+ and calcium contents in slow-twitch and fast-twitch muscles were compared. Whereas intermittent stimulation for 24 h at 20 Hz caused only minor changes in soleus (SOL), a considerable loss of K+ (around 24%) and gain of Na+ (around 84%) was observed in extensor digitorum longus (EDL) and tibialis anterior (TA) muscles. These changes could be detected within 0.5 h and a plateau was maintained from 2 to 24 h. Total calcium content increased progressively, reaching values 245 and 382% above the control level in EDL and TA muscle, respectively, after 24 h of 20 Hz stimulation. Whereas the Na+ and K+ content recovered within a few hours, calcium content did not return towards control level until after 48 h of rest. In a pilot study performed with continuous stimulation at 10 Hz, the changes in Na+ and K+ contents in SOL, EDL and TA muscle were comparable to those at 20 Hz. The concentration of the Na(+)-K+ pumps was highest in the fast-twitch EDL and TA muscles and was unaffected by 10 Hz stimulation. It is concluded that a stimulation pattern leading to a rise in intracellular Na+ and a loss of K+ may cause a marked accumulation of calcium. These events seem to be related to insufficient activation of the Na-K+ pump rather than to variations in the total Na(+)-K+ pump capacity.

Charnockitic alteration: evidence for CO2 infiltration in granulite facies metamorphism

Charnockitic alteration (arrested orthopyroxene formation in biotite‐ and amphibole‐bearing rocks) occurs in high‐grade terranes of all ages. Three criteria are used to show that this alteration was produced in many locations by a migrating fluid phase: (i) diffuseness of the alteration—the alteration zones are often quite unlike discrete migmatitic veins; (ii) relation to deformation—most occurrences show alteration closely associated with warping of foliation or dilation cracks; (iii) open‐system alteration—whilst some occurrences represent nearly isochemical alteration, slight changes in bulk composition, often loss of mafic constituents and gain of Na and Si, are evident in detailed mass‐balance analysis. Y and sometimes Rb are characteristically depleted. Partial melting sometimes accompanied volatile infiltration, as evidenced by more discrete veins and euhedral orthopyroxene. It is quite unlikely, however, that open‐system alteration was produced by escape of viscous quartzo‐feldspathic melts. Pervasive migration of low‐T lamprophyric (mafic–alkaline, CO2‐charged) interstitial liquids is a possibility by virtue of their extreme fluidity, but CO2 infiltration was needed to generate these liquids. Vapour‐deficient dehydration melting is another feasible mechanism of orthopyroxene formation which may have operated in conjunction with CO2 infiltration.Characteristic development of charnockitic alteration in some prograde amphibolite to granulite facies transitions, as in the Dharwar Craton of South India, suggests that the alteration is a fundamental feature of the granulite facies metamorphism, implying active and causal participation of migrating fluids. In other high‐grade terranes like the Adirondack Mountains of New York, this kind of alteration is rare, and fluid action does not seem to have been important in the metamorphism.A vapour phase participating in charnockitic metamorphism was necessarily one of relatively low H2O, therefore presumably rich in CO2. Consideration of possible large CO2 sources leads to the conclusion that emanations from volatile‐rich basalts emplaced in the lower crust are the most probable source of charnockitizing fluids. The ultimate source would therefore be enriched subcontinental lithosphere or asthenosphere. The Rb‐depleted pyroxene gneiss (charnockitic) terranes may be characteristic of zones of large‐scale transcurrent or oblique‐motion faults which tap such great depths.