Living with water stress: evolution of osmolyte systems.

Striking convergent evolution is found in the properties of the organic osmotic solute (osmolyte) systems observed in bacteria, plants, and animals. Polyhydric alcohols, free amino acids and their derivatives, and combinations of urea and methylamines are the three types of osmolyte systems found in all water-stressed organisms except the halobacteria. The selective advantages of the organic osmolyte systems are, first, a compatibility with macromolecular structure and function at high or variable (or both) osmolyte concentrations, and, second, greatly reduced needs for modifying proteins to function in concentrated intracellular solutions. Osmolyte compatibility is proposed to result from the absence of osmolyte interactions with substrates and cofactors, and the nonperturbing or favorable effects of osmolytes on macromolecular-solvent interactions.

Stoichiometry and ion dependencies of the intracellular-pH-regulating mechanism in squid giant axons.

The ion transport system responsible for intracellular pH (pHi) regulation in squid giant axons was examined in experiments with pH-sensitive microelectrodes and isotopic fluxes of Na+ and Cl-. In one study, axons were acid-loaded and the rate of the subsequent pHi recovery was used to calculate the acid extrusion rate. There was an absolute dependence of acid extrusion on external Na+, external HCO-3 (at constant pH), and internal Cl-. Furthermore, the dependence of the acid extrusion rate on each of these three parameters was described by Michaelis-Menten kinetics. Acid extrusion was stimulated by an acid pHi, required internal ATP, and was blocked by external 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate (SITS). Under a standard set of conditions (i.e., [HCO-3]o = 12 mM, pHo = 8.00, [Na+]o = 425 mM, [Cl-]i = 150 mM, [ATP]i = 4 mM, pHi = 6.5, and 16 degrees C), the mean acid extrusion rate was 7.5 pmol X cm-2 X s-1. In a second study under the above standard conditions, the unidirectional Na+ efflux (measured with 22Na) mediated by the pHi-regulating system was found to be approximately 0, whereas the mean influx was about 3.4 pmol X cm-2 X s-1. This net influx required external HCO-3, internal Cl-, and acid pHi, internal ATP, and was blocked by SITS. In the final series of experiments under the above standard conditions, the unidirectional Cl- influx (measured with 36Cl) mediated by the pHi-regulating system was found to be approximately 0, whereas the mean efflux was approximately 3.9 pmol X cm-2 X s-1. This net efflux required external HCO-3, external Na+, an acid pHi, internal ATP, and was blocked by SITS. We conclude that the pHi-regulating system mediates the obligate net influx of HCO-3 (or equivalent species) and Na+ and the net efflux of Cl- in the stoichiometry of 2:1:1. The transport system is stimulated by intracellular acid loads, requires ATP, and is blocked by SITS.

Membrane and communication properties of tissue cultured human lens epithelial cells.

Explant cultures were established from capsule/epithelium preparations from both normal and cataractous lenses to investigate properties of human lens epithelial cells. The cultured cells were found to have similar membrane potentials to whole human lenses and isolated epithelia, and similar free ionic concentrations of potassium, sodium, and calcium (131 mM, 17 mM and 0.8 microM respectively) to whole human lenses. The free ionic concentrations were measured in both cases using neutral resin-filled electrodes. Cellular communication was investigated using electrical (two internal microelectrodes) and dye injection techniques. The electrical resistance of a confluent cell monolayer was approximately 4 M omega when the voltage measuring and current passing microelectrodes were in neighbouring cells or several cell diameters apart. Additionally, Lucifer Yellow dye injected into one cell spread rapidly over a wide area of cells. The cells thus appear to be extremely well coupled. Electrical communication could be disrupted by internal acidification (following exposure to 100% CO2), exposure to 1 mM octanol and by membrane depolarization. In the latter case the blockade was only partial. All uncoupling methods proved to be reversible. The diffusion of Lucifer Yellow dye was also inhibited by internal acidification and exposure to octanol. The sensitivity of both dye and electrical coupling to internal acidification and exposure to octanol is similar to that observed in hepatocytes and other tissues, whereas the effect on cell communication induced by changing the resting potential appears to occur only in a few cell types such as those of embryonic origin.

Cardiovascular effects of contrast agents.

Iodinated radiologic contrast agents should, ideally, passively provide radiographic contrast in blood vessels and soft tissues but have no effects on physiology, hematology or biochemistry. In practice, no such agent exists although the nonionics most closely approach the ideal. All agents have a bewildering variety of effects on the cardiovascular system, which, in turn, engender a variety of neurogenic and humoral responses. The mechanisms underpinning these many phenomena appear to involve hyperosmolality, molecular structure, ionic content and balance. This review presents the current state of knowledge of these effects and their mechanisms, with emphasis on the differences between conventional ionic and more inert nonionic agents.

Molecular charge effects on the protein permeability of the guinea-pig placenta.

The effect of molecular charge on protein permeability of the dually perfused guinea-pig placenta has been investigated by measurement of the permeability surface area products (PS) and observation of the ultrastructural localization of cationic horseradish peroxidase (cHRP) and anionic horseradish peroxidase (aHRP) molecules. Steady-state PS calculated from the experimental data was 0.032 +/- 0.0045 and 0.0045 +/- 0.0008 ml min-1 (mean +/- s.e.m.) for cHRP and aHRP respectively (P less than 0.01). The PS for a diffusional marker, 51Cr-ethylenediaminetetraacetic acid, showed no significant difference between the two groups. Ultrastructurally, placentae perfused with cHRP showed fewer microvilli and a dilated interstitial space compared with placentae perfused with aHRP; large vacuoles were also found in the syncytiotrophoblast in the former but not in the latter tissue. Reaction product in placentae perfused with cHRP was localized to the maternal-facing plasma membrane of the syncytiotrophoblast, in vacuoles and vesicles of the syncytiotrophoblast, and also in the basement membrane of the interstitial space, whereas placentae perfused with aHRP only had reaction product in vesicles in the syncytiotrophoblast. These results suggest that anionic sites in the guinea-pig placenta affect its permeability to charged proteins, cationic molecules inducing structural changes associated with increased permeability.

Spinal cord sodium, potassium, calcium, and water concentration changes in rats after graded contusion injury.

Spinal cord Na, K, Ca, and H2O changes were measured 6 h after graded contusion injuries in 40 Sprague-Dawley rats. A 10 g weight was dropped 1.25 cm (n = 6), 2.5 cm (n = 7), 5.0 cm (n = 6), or 7.5 cm (n = 7) onto the thoracic spinal cord of 26 rats. An additional 10 rats served as laminectomy controls and 4 rats were unoperated controls. At 6 h after surgery or injury, the spinal cords were rapidly cut into 4 mm segments, weighed to obtain tissue wet weights (W), dried for 14-16 h at 97 degrees C in a vacuum oven (30 mmHg), and reweighed for tissue dry weights (D). Water concentrations ([H2O]d) were estimated from (W-D)/D in units of ml/g D. Ionic concentrations ([Na]d, [K]d, and [Ca]d) of the tissue samples were measured by atomic absorption spectroscopy with units of mumol/g D. Ionic shifts (delta [Na]d, delta [K]d, delta [Ca]d) were calculated by subtracting laminectomy control values from those measured in injured cords. Laminectomy alone significantly increased [Na]d and [H2O]d compared to unoperated controls. Mean +/- standard deviations of [H2O]d, [Na]d, [K]d, and [Ca]d were, respectively, 1.95 +/- 0.07, 182.6 +/- 5.9, 277.2 +/- 11.8, and 12.1 +/- 1.4 in unoperated controls; 2.12 +/- 0.08, 238.6 +/- 9.2, 277.8 +/- 9.2, and 11.7 +/- 1.1 in laminectomy controls. At the impact site, [K]d fell by 14-37% and [H2O]d rose by 14-24%, [Na]d by 13-64%, and [Ca]d by 65-137% of laminectomy control values. delta [Na]d, delta [K]d, and delta [Ca]d correlated linearly with impact velocities; [Ca]d increased by 1.0% per cm/sec (r = 0.995, p less than 0.005), [Na]d increased 0.67% per cm/sec (r = 0.950, p less than 0.01), and [K]d decreased 0.34% per cm/sec (r = 0.964, p less than 0.01). Neither delta [H2O] nor delta [Na]d + delta [K]d consistently predicted impact velocity. [Na]d + [K]d correlated with [H2O]d with a slope of 177.4 mumol/ml (r = 0.697, p less than 0.005). Since Na and K constitute greater than 95% of tissue inorganic ions, the slope approximates net ionic shift per ml of water entry or the ionic osmolarity of edema fluid. These results indicate that increasing contusions produce graded ionic shifts and that edema does not predict contusion severity. These data support our hypothesis that net ionic shifts cause edema in injured spinal cords.

Electrophysiological and biochemical characterization of a continuous human astrocytoma cell line with many properties of well-differentiated astrocytes.

Astrocytes comprise about 25% of the cellular volume of the brain, and their main function is to maintain homeostasis of the neuronal environment. These cells are commonly identified on the basis of their membrane electrical properties and the presence of specific proteins. We have characterized the human astrocytoma cell line designated UC-11MG and have shown these cells have many of the traits of differentiated astrocytes. Many of the UC-11MG cells have a large resting membrane potential, averaging -74 mV. The slope of the Em vs log [K]o cuve was 58.5 mV per decade [K]o. The cells were inexcitable when exposed to brief depolarizing current pulses. The astrocytoma traits are virtually identical to those previously reported for normal astrocytes. The astrocytoma cells also express glutamine synthetase activity which is considered specific to astrocytes among brain cells. Previous work had also demonstrated the presence of other astrocyte markers glial fibrillary acidic protein and S-100 protein in the UC-11MG cells. The steady-state ion transport properties of Na+, Cl-, and K+ were also characterized in these cells, and the rates of efflux were found to be similar to those in other astrocytes, with the major difference being the presence of a second kinetic compartment in the UC-11MG cells. From this work, we conclude that the UC-11MG cell line displays prominent features associated with differentiated astrocytes, and may provide an excellent model system for the study of human astrocytes.

The effect of therapeutic ultrasound on electrophysiological parameters of frog skin.

There are two groups of mechanisms through which ultrasound can affect biological systems, those of thermal origin and others of nonthermal origin. Since in almost every therapeutic application of ultrasound, movement of ions across cellular membranes is involved, it becomes important to study the effect of ultrasound on active and passive ionic conductance. In order to differentiate between thermal and nonthermal effects, a study was conducted on model systems in which the effect of temperature is known. The well-known sodium transporting epithelium, the epidermis of abdominal frog skin, was investigated and the effect of therapeutic ultrasound on its electrophysiological properties was determined. It was found that under open circuit conditions, irradiation of the skin with 1 MHz cw (60-480 mW/cm2) ultrasound caused a significant decrease (5-50%, depending on the applied power) in the transepithelial potential and resistance at room temperature (20-22 degrees C). Under short circuit conditions, also at room temperature, there was an increase in total ionic conductance (20-250%, depending on the applied power) and a decrease in the net actively transported current, measured as the short circuit current. These effects are reversible within the range of powers used. Furthermore, it was found that the magnitude of the observed changes was strongly dependent on the perfusion rate and the gas content of the bathing medium. The effect of ultrasound diminished in the presence of CO2 and was enhanced with faster perfusion rates. Pulsed ultrasound delivered at the same energy (Isata) as that of cw caused a significantly larger effect. At lower temperatures (12-14 degrees C) the effect of ultrasound was reduced. Analysis of the data reveals that the effects of ultrasound on ion transport reported here are not primarily of thermal origin but are probably due to cavitation and related effects, such as microsteaming.

Non-invasive monitoring of ionic current flow during development by SQUID magnetometry.

The ionic currents flowing in developing organisms produce weak magnetic fields that can be detected using SQUID magnetometers. The method is non-invasive and dc recording is possible. To date SQUID magnetometers have mainly been used in human studies. The features of the technique are described and the prospects of extending its use to developmental studies are discussed. Feasible instrumental specifications are indicated. A recent SQUID magnetometer investigation of ionic current flow in the developing chick in ovo is summarised as an illustration of the magnetometer method. The paper as a whole argues that magnetometry is a useful alternative or adjunct to electrode-based experiments on the electrophysiology of developing organisms.

Applicability of available ion-selective liquid-membrane microelectrodes to intracellular ion-activity measurements.

The equations that govern the behavior of ion-selective electrodes are evaluated for the composition ranges of Li+, Na+, K+, Mg2+, Ca2+, Cl-, and HCO-3 encountered in living cells. Various cause-effect relations are detailed, especially those pertaining to calibration procedures. The relation of the accuracy of measurement to the expected signal range, particularly in connection with the uncertainty of interpolation, is emphasized. Relevant numerical parameters are given for all ISE's discussed, the selectivity characteristics being those of the most advanced membrane systems. Figures are provided for the calibration curves in the vicinity of the typical cytosol composition.

Ionic currents in morphogenesis.

Morphogenetic fields must be generated by mechanisms based on known physical forces which include gravitational forces, mechanical forces, electrical forces, or some combination of these. While it is unrealistic to expect a single force, such as a voltage gradient, to be the sole cause of a morphogenetic event, spatial and temporal information about the electrical fields and ion concentration gradients in and around a cell or embryo undergoing morphogenesis can take us one step further toward understanding the entire morphogenetic mechanism. This is especially true because one of the handful of identified morphogens is Ca2+, an ion that will not only generate a current as it moves, but which is known to directly influence the plasma membrane's permeability to other ions, leading to other transcellular currents. It would be expected that movements of this morphogen across the plasma membrane might generate ionic currents and gradients of both electrical potential and intracellular concentration. Such ionic currents have been found to be integral components of the morphogenetic mechanism in some cases and only secondary components in other cases. My goal in this review is to discuss examples of both of these levels of involvement that have resulted from investigations conducted during the past several years, and to point to areas that are ripe for future investigation. This will include the history and theory of ionic current measurements, and a discussion of examples in both plant and animal systems in which ionic currents and intracellular concentration gradients are integral components of morphogenesis as well as cases in which they play only a secondary role. By far the strongest cases for a direct role of ionic currents in morphogenesis is the polarizing fucoid egg where the current is carried in part by Ca2+ and generates an intracellular concentration gradient of this ion that orients the outgrowth, and the insect follicle in which an intracellular voltage gradient is responsible for the polarized transport from nurse cell to oocyte. However, in most of the systems studied, the experiments to determine if the observed ionic currents are directly involved in the morphogenetic mechanism are yet to be done. Our experience with the fucoid egg and the fungal hypha of Achlya suggest that it is the change in the intracellular ion concentration resulting from the ionic current that is critical for morphogenesis.

Design of ionophores for ion-selective microsensors.

Requirements for a reliable use of liquid membrane microelectrodes are discussed in terms of stability, response time, and lifetime on the basis of membrane technological considerations. The selectivity of H+, Li+, Na+, K+, Mg2+, Ca2+, and Cl- microelectrodes is critically evaluated using the Nikolskii-Eisenman formalism. Recent progress in the design of new ionophores is presented. A novel neutral carrier-based Ca2+-selective microelectrode with a detection limit of about 5 X 10(-10) M Ca2+ at a background of 125 mM K+ has been realized. An neutral carrier-based microelectrode for H+ with extended pH range of the sample solution is now available. Promising developments in the field of Li+-, Mg2+-, and Cl--selective ionophores are discussed.

Inorganic ion pairs in physiology: significance and quantitation.

1. Body fluids contain ion pairs such as NaC10, CaCl+, CaHCO+3 and MgSO04. 2. Ion pairs must often be considered in the quantitation of acid-base, solubility, Donnan and other equilibria. 3. Fluxes of ion pairs, such as NaCO-3 and perhaps NaSO-4 and NaCl0, may sometimes contribute significantly to total ion fluxes across cell membranes. 4. Dissociation constants for ion pairs are discussed, but values appropriate to body fluids are often uncertain.