Interaction of hemolysis and genotype on ionized calcium in bile of mice with hemolysis-induced gallstones.

In this study, we used an ion-selective membrane electrode to measure ionized calcium in hepatic bile of control +/+ mice and nb/nb mice with hereditary hemolytic anemia. We found that biliary concentrations of ionized, bound, and total calcium were significantly higher (p less than 0.001) and magnesium was significantly lower (p less than 0.001) in nb/nb mice than in control +/+ mice. To separate the hemolytic process from genotypic influences, we transplanted genetically defective bone marrow from nb/nb mice into histocompatible nonhemolytic recipients (W/Wv). After successful engraftment, transplanted W/Wv mice had significantly higher biliary concentrations of ionized calcium than their untreated W/Wv counterparts (p less than 0.001); but bound and total calcium and magnesium concentrations were not different from untreated W/Wv controls. When compared with nb/nb mice, transplanted W/Wv mice had lower ionized calcium (p less than 0.001) and higher bound calcium concentrations (p less than 0.001) in their biles. These data indicate that ionized calcium in hepatic bile is significantly influenced by genotypic factors and subsequently increased in chronic hemolysis; and further, that increased ionized calcium in bile of mice with hemolysis is a risk factor, but of limited predictive value for hemolysis-induced gallstone formation.

Calcium carrying system in the giant muscle fibre of the barnacle species, Balanus nubilus.

1. Single barnacle muscle fibres from Balanus nubilus were internally perfused with an isotonic solution containing 180 mM-tetraethylammonium acetate and the effects of Ca concentration in the external solution on the voltage-clamp currents, especially the initial inward current, were examined.2. Muscle fibre in external solution containing no added Ca (concentration of Ca < 10(-5)M) gave a current-voltage curve that showed a small inward current followed by a small outward current. An identical curve was obtained when the chloride in the external solution was replaced by acetate.3. The peak inward current increased with increase in Ca concentration in the external solution; but the increase in peak current for equal increments of Ca concentration was reduced and attained saturation conforming to an adsorption regime which can be described by the Langmuir equation.4. A double reciprocal plot of peak inward current due to Ca as a function of Ca concentration gave values of 20.7 mM and 200% (the current due to 20 mM-Ca normalized to 100%) for the dissociation constant and the maximum current respectively. These values were found to depend on the concentration of Mg used in the external solutions.5. The peak inward Ca currents measured at two Ca concentrations as a function of pH were unaffected in the pH range 6.5-8.8; however when the pH was reduced below 6.5, the currents were depressed.

Some electrical properties of the membrane of the barnacle muscle fibers under internal perfusion.

Intracellular perfusion technique has been applied to the muscle fibers of the barnacle species, Balanus nubilus. In these fibers, generation and the form of the calcium spike was governed by the frequency of stimulation and intra- and extracellular calcium concentrations. Voltage-clamp experiments showed that the magnitude of the potassium outward current was controlled by the intracellular calcium concentration whose increase, nearly 10(3)-fold, raised the resting membrane conductance and the outward potassium current. On the other hand, application of 10 mM zinc ions inside the muscle fiber had no effect on either the resting potential or the outward potassium current but suppressed the early inward calcium current. Similarly, the inward calcium current was decreased by low concentration of sodium ions in the extracellular fluid only when its ionic strength was made low by substituting sucrose for the sodium salt. Measurement of outward current with the muscle fiber in calcium-free ASW solution and intracellularly perfused with several cationic solutions established the selectivity sequence TEA less than Cs less than Li less than Tris less than Rb less than Na less than K for the potassium channel.

Surface charges on membranes.

The equations of membrane potential developed by Kobatake and coworkers have been applied to the literature data on the resting membrane potential of the crayfish and Myxicola axons to derive values for the surface charge density present on the axon membranes. Some shortcomings of the method are briefly discussed. The value for the surface charge density derived for the squid axon membrane agreed with a similar value derived from measurements of shifts in Na and/or potassium conductance-voltage relations following changes in the concentration of calcium in the solutions bathing the axons.

Effects of pH and ionic strength on the potassium system in the internally perfused giant barnacle muscle fibre.

Membrane potentials in single barnacle muscle fibres of Megabalanus psittacus, internally perfused with 200 mM K-acetate (KAc) solution of pH 7.5 were in the range --53 to --60 mV. These were followed as a function of pH of the external chloride saline. Decrease of pH of the Cl-saline from 8.5 to 3.5 hyperpolarized the membrane reversibly by about 8 mV. On further decrease of pH to 3.0, a transient hyperpolarization (from --60 to --65 mV) followed by a sudden and irreversible drop of potential to --40 mV was observed. Replacement of the external Cl-saline by acetate saline in the pH range 8.5 to 6.5 had no effect on the membrane potential. Further decrease of pH to 5.0 brought about an irreversible reduction of membrane potential. For fibres bathing in Cl- or Ac-saline at pH 7.5 when the internal pH was changed to 5.5, a transient hyperpolarization in Cl-saline and a sustained hyperpolarization in Ac-saline were observed. Further studies of membrane potential changing the concentration of external K, keeping the concentration of Cl or Ac constant, and changing the concentration of Cl or Ac, keeping the concentration of K constant, at different internal and external pHs showed that as the pH was reduced the membrane became more permeable to anions, Cl being more permeable than Ac. Experiments in which the membrane potential was controlled showed that when the internal pH of 200 mM KAc solution was reduced from 7.5 to 6.5 or raised to 8.5, the changes in outward K currents at various depolarizations were negligible. However, when the pH was reduced to 5.5 or 5.0, there was a progressive decrease in the outward K currents. The leakage currents in all these cases were relatively small. Use of high ionic strength solution of 580 mM KAc internally protected the K system in that when the pH was lowered the depression of the outward K current was smaller than that observed at a corresponding pH when the internal solution was 200 mM KAc. Use of low ionic strength solution of 50 mM KAc had the opposite effect. The results have been explained in terms of the ion exchange theory by postulating that the membrane has fixed amphoteric groups.

Estimation of surface charges in some biological membranes.

The resting membrane potential data existing in the literature for the giant axon of the squid, frog muscle and barnacle muscle have been analyzed from the standpoint of the theory of membrane potential due to Kobatake and co-workers. The average values derived for the effective charge density phi chi (where phi is a constant, 0 less than phi less than 1, and represents the fraction of counterions that are free, and chi is the stoichiometric charge density in the membrane) present on the different biomembranes existing in their normal ionic environment are 0.3, 0.325 and 0.17 M for the squid axon, frog and barnacle muscles, respectively. On the assumption that the values of phi are 0.4 and 0.2 for nerve and muscle membranes, respectively, values of 0.75, 1.62 and 0.85 M have been derived for the stoichiometric charge density (chi) present in the respective biological membranes. These correspond to 1 negative charge per 222, 103 and 195 A2 of the membrane area of the squid axon, frog and barnacle muscles, respectively.

Effects of anions and cations on the resting membrane potential of internally perfused barnacle muscle fibres.

1. Single barnacle muscle fibres from Megabalanus psittacus (Darwin) were internally perfused with a number of K salt solutions (200 mM) which were made isotonic to the barnacle saline with sucrose.2. 200 mM-K acetate solution, in general, was found to be more effective than other solutions of K salts in generating and maintaining stable resting membrane potential of -56.0 +/- 0.7 mV (all potentials are referred to the external solutions as ground). The various K salts, on the basis of the magnitude of the resting potential they generated in the muscle fibres, followed the sequence, acetate > isethionate > aspartate > glutamate > fluoride > monohydrogen phosphate > succinate > citrate > sulphate > oxalate > iodobenzoate > ferrocyanide > chlorate > nitrate > chloride > thiocyanate > iodide > bromide > cyanide.3. The resting potential in muscle fibres perfused with solutions of acetate, aspartate and glutamate increased linearly with the logarithm of the K concentration (slope = 30.4 mV for K acetate and 27.4 for K aspartate and glutamate) when the ionic strength of the solutions was progressively increased from 50 to 650 mM. On the other hand, similar increase of ionic strength beyond 200 mM of solutions of K isethionate, fluoride, monohydrogen phosphate, succinate and citrate depolarized the muscle fibres.4. Perfusion of acetate solutions of other alkali metal ions gave low values for the resting potential and followed the sequence K > Na > Rb > Li > Cs. Also NH(4) and Tris ions gave low values for the resting potential which underwent oscillations associated with the twitching of the fibre and occasionally became positive in value (action potential).5. Addition of tetraethyl ammonium chloride (TEA-Cl), 20-100 mM, to K acetate solutions (200 mM) depolarized the fibre membrane and the consequent reduction of resting potential varied linearly with the logarithm of TEA concentration.6. Replacement of chloride ion by acetate or isethionate in the external solution did not change significantly the resting potential although the values were consistently lower by about 2 mV.7. Complete elimination of K in the external solution and reduction of its ionic strength using sucrose depolarized the muscle fibres by about 27 mV when Na was changed from 475 to 1 mM. Under these conditions, external solutions completely in acetate form gave resting potentials which were more positive than those observed in completely chloride solutions by 6-8 mV.8. Replacement of Na by Li, Tris, choline, tetramethyl or tetraethyl ammonium ion in the external solution made the values of the resting potential more positive (depolarization). Similarly increasing the concentration of K (or Cs or Rb in place of K) by correspondingly decreasing the concentration of Na in the outside solution depolarized the fibres and the resting potential became zero at a concentration of 280 mM (or 308 or 1500 mM for Rb or Cs, respectively) on extrapolation.

Studies with composite membranes. 3. Measurement of water permeability.

The permeability of tritiated water (THO) across simple and layer-type composite membranes of collodion containing different amounts of polystyrenesulfonic acid has been measured and corrected for the effects of aqueous stationary layers present at the membrane-solution interfaces. It was found that the water permeabilities in the two opposite directions across the composite membranes were different, whereas they were the same across simple membranes. The theoretical permeability value for the composite membrane (formed by putting one simple membrane on top of another simple membrane of increasing charge density and gently pressing them together), calculated from the values due to simple membranes, was found to be always greater than the two measured values. It was shown that the aqueous layers trapped between membranes were not responsible for the low measured values. The factor causing this was ascribed to the mechanism which produced rectification of water flow in the composite membranes. Establishment of the THO concentration profile in the layered membranes showed that accumulation and depletion of THO in the membrane phase when the THO was flowing from the high charge density side to the low charge density side and vice versa, respectively, were responsible for the unequal flows observed across the composite membrane in the two directions.

Studies with composite membranes. Preparation and measurement impedances.

Simple and composite membranes have been prepared from 2% collodion solutions containing different amounts of polystyrenesulfonic acid (PSSA). Various membrane parameters such as water content, electrolyte uptake, exchange capacity, and permselectivity of these membranes have been determined. The resistance and capacitance of simple membranes have been measured as functions of both external electrolyte concentration and internal fixed charge density. The impedance characteristics of composite membranes also have been determined and discussed in terms of the resistance and capacitance characteristics of simple membranes from which the composite structures have been formed.

Studies with composite membranes. II. Measurement of asymmetric properties.

Electrical potentials arising across composite membranes when they separate the same concentration of a (1:1) electrolyte or electrolytes have been measured. These potentials have been shown to arise from differences in the transport number of counterions contacting the two faces of the membrane which contained in its body a high concentration of electrolyte and polyelectrolyte. When the concentration of this trapped electrolyte or polyelectrolyte is low, the asymmetry potentials are small. Although measurements of current-voltage relations provided evidence for the existence of asymmetry between the two faces of the membrane, osmotic flow of water in either direction across the membrane and the salt flow in the two directions were symmetrical. These solvent and solute flux measurements lasted more than 30 hr. Short-term (about 4 hr) flux measurements, however, using tritiated water (THO), gave flows which were different in the two directions. Similarly, the salt flows measured using (22)Na isotope were different in the two directions. The usefulness of the present system as a model to use for studies concerned with carrier transport problems in biology has been pointed out.