Hepatocyte water volume and potassium activity during hypotonic stress

Hepatocytes exhibit a regulatory volume decrease (RVD) during hypotonic shock, which comprises loss of intracellular K⁺ and Cl^– accompanied by hyperpolarization of transmembrane potential (V _m ) due to an increase in membrane K⁺ conductance, (G _K). To examine hepatocyte K⁺ homeostasis during RVD, double-barrel, K⁺-selective microelectrodes were used to measure changes in steady-state intracellular K⁺ activity (a _K ⁱ ) and V _m during hyposmotic stress. Cell water volume change was evaluated by measuring changes in intracellular tetramethylammonium (TMA⁺). Liver slices were superfused with modified Krebs physiological salt solution. Hyposmolality (0.8×300 mosm) was created by a 50 mm step-decrease of external sucrose concentration. Hepatocyte V _m hyperpolarized by 19 mV from –27 ± 1 to –46 ± 1 mV and a _K ⁱ decreased by 14% from 91 ± 4 to 78 ± 4 mm when slices were exposed to hyposmotic stress for 4–5 min. Both V _m and a _K ⁱ returned to control level after restoring isosmotic solution. In paired measurements, hypotonic stress induced similar changes in V _m and a _K ⁱ both control and added ouabain (1 mm) conditions, and these values returned to their control level after the osmotic stress. In another paired measurement, hypotonic shock first induced an 18-mV increase in V _m and a 15% decrease in a _K ⁱ in control condition. After loading hepatocytes with TMA⁺, the same hypotonic shock induced a 14-mV increase in V _m and a 14% decrease in a _TMA ⁱ . This accounted for a 17% increase of intracellular water volume, which was identical to the cell water volume change obtained when a _K ⁱ was used as the marker. Nonetheless, hyposmotic stress-induced changes in V _m and a _K ⁱ were blocked partly by Ba²⁺ (2 mm). We conclude that (i) hepatocyte V _m increases and a _K ⁱ decreases during hypotonic shock; (ii) the changes in hepatocyte V _m and a _K ⁱ during and after hypotonic shock are independent of the Na⁺-K⁺ pump; (iii) the decrease in a _K ⁱ during hypotonic stress results principally from hepatocyte swelling.This work was supported by grant AA-08867 from the Alcohol, Drug Abuse, and Mental Health Association.     

Soil solute concentration and water uptake by single lupin and radish plant roots

Application of computer assisted tomography to gamma and X-ray attenuation measurements and Na⁺-LIX microelectrodes were used to determine the spatial distributions of soil water content and Na⁺ concentrations respectively near single roots of eighteen day old lupin and radish plants. These quantities were monitored at root depths of 3, 6 and 9 cm and at zero, 2, 4, 6, and 8 hour intervals from the diurnal commencement of transpiration. The plants were subjected to two levels of transpirational demand and five Na⁺ soil solution concentration levels. Water extraction rates for the lupin and radish roots increased continuously with time but were substantially reduced with increasing Na⁺ concentration in the treatment. Water uptake was uniform along the length of the essentially constant diameter lupin roots but decreased along the tapering radish roots as the diameter and hence the surface area per unit length of the roots decreased. The accumulation of Na⁺ at the root surfaces of both plants increased gradually with time in a near linear fashion and was slightly higher under the higher transpiration demand. These increases were not exponential as would be expected with non-absorption by the roots and this is considered to be due to back diffusion at the relatively high water contents used. At these water contents matric potentials had a much smaller influence on transpiration than osmotic potentials. The relationships between leaf water potentials (Ψ₁) and osmotic potentials at the root surfaces were linear with the decreases in Ψ₁ almost exactly reflecting the decreases in Ψ_π indicating rapid plant adjustment. Leaf water potentials decreased progressively with time and the relationships between leaf water potential and the transpiration rate were also linear supporting the suggestion of constant plant resistances at any given concentration.     

Presynaptic Dopamine Dynamics in Striatal Brain Slices with Fast-scan Cyclic Voltammetry

Extensive research has focused on the neurotransmitter dopamine because of its importance in the mechanism of action of drugs of abuse (e.g. cocaine and amphetamine), the role it plays in psychiatric illnesses (e.g. schizophrenia and Attention Deficit Hyperactivity Disorder), and its involvement in degenerative disorders like Parkinson''s and Huntington''s disease. Under normal physiological conditions, dopamine is known to regulate locomotor activity, cognition, learning, emotional affect, and neuroendocrine hormone secretion. One of the largest densities of dopamine neurons is within the striatum, which can be divided in two distinct neuroanatomical regions known as the nucleus accumbens and the caudate-putamen. The objective is to illustrate a general protocol for slice fast-scan cyclic voltammetry (FSCV) within the mouse striatum. FSCV is a well-defined electrochemical technique providing the opportunity to measure dopamine release and uptake in real time in discrete brain regions. Carbon fiber microelectrodes (diameter of ~ 7 μm) are used in FSCV to detect dopamine oxidation. The analytical advantage of using FSCV to detect dopamine is its enhanced temporal resolution of 100 milliseconds and spatial resolution of less than ten microns, providing complementary information to in vivo microdialysis.     

Carbonic anhydrase generates a pH gradient in Bombyx mori silk glands

Silk is a protein of interest to both biological and industrial sciences. The silkworm, Bombyx mori, forms this protein into strong threads starting from soluble silk proteins using a number of biochemical and physical cues to allow the transition from liquid to fibrous silk. A pH gradient has been measured along the gland, but the methodology employed was not able to precisely determine the pH at specific regions of interest in the silk gland. Furthermore, the physiological mechanisms responsible for the generation of this pH gradient are unknown.In this study, concentric ion selective microelectrodes were used to determine the luminal pH of B. mori silk glands. A gradient from pH 8.2 to 7.2 was measured in the posterior silk gland, with a pH 7 throughout the middle silk gland, and a gradient from pH 6.8 to 6.2 in the beginning of the anterior silk gland where silk processing into fibers occurs. The small diameter of the most anterior region of the anterior silk gland prevented microelectrode access in this region. Using a histochemical method, the presence of active carbonic anhydrase was identified in the funnel and anterior silk gland of fifth instar larvae. The observed pH gradient collapsed upon addition of the carbonic anhydrase inhibitor methazolamide, confirming an essential role for this enzyme in pH regulation in the B. mori silk gland. Plastic embedding of whole silk glands allowed clear visualization of the morphology, including the identification of four distinct epithelial cell types in the gland and allowed correlations between silk gland morphology and silk stages of assembly related to the pH gradient.B. mori silk glands have four different epithelial cell types, one of which produces carbonic anhydrase. Carbonic anhydrase is necessary for the mechanism that generates an intraluminal pH gradient, which likely regulates the assembly of silk proteins and then the formation of fibers from soluble silk proteins. These new insights into native silk formation may lead to a more efficient production of artificial or regenerated silkworm silk fibers.     

Photosynthesis,Respiration and Exopolymer Calcium-Binding in Biofilm Calcification (Westerhöfer and Deinschwanger Creek,Germany)

The impact of microbial activity on biofilm calcification in aquatic environments is still a matter of debate, especially in settings where ambient water has high CaCO₃ mineral supersaturation. In this study, biofilms of two CO₂-degassing karst-water creeks in Germany, which attain high calcite supersaturation during their course downstream, were investigated with regard to water chemistry of the biofilm microenvironment. The biofilms mainly consisted of filamentous cyanobacteria (Phormidium morphotype) and heterotrophic bacteria (including sulfate-reducing bacteria), which affect the microenvironment and produce acidic exopolymers. In situ and ex situ microelectrode measurements showed that a strong pH increase, coupled with Ca^{2 +} consumption, occurred in light conditions at the biofilm surface, while the opposite occurred in the dark. Calcite supersaturation at the biofilm surface, calculated from ex situ Ca^{2 +} and CO₃ ^2? microelectrode measurements, showed that photosynthesis resulted in high omega values during illumination, while respiration slightly lowered supersaturation values in the dark, compared to values in the water column. Dissociation calculation demonstrated that the potential amount of Ca^{2 +} binding by exopolymers would be insufficient to explain the Ca^{2 +} loss observed, although Ca^{2 +} complexation to exopolymers might be crucial for calcite nucleation. No spontaneous precipitation occurred on biofilm-free limestone substrates under the same condition, regardless of high supersaturation. These facts indicate that photosynthesis is a crucial mechanism to overcome the kinetic barrier for CaCO₃ precipitation, even in highly supersaturated settings.     

Carbonate Mineralogy Along a Biogeochemical Gradient in Recent Lacustrine Sediments of Gallocanta Lake (Spain)

Three sedimentary subenvironments, palustrine (GP), marginal lacustrine (GML) and central lacustrine (GCL), were compared regarding water chemistry and microbial activity in order to explain the differences in the carbonate mineralogical composition of the upper sediment layer in Gallocanta Lake, a shallow hypersaline environment in Northeastern Spain. Horizontal heterogeneity was considerable, salinity ranged from 5 to 116 (‰) for the GP and GCL subenvironments respectively. Sulfate, Mg ^{2 +} , and Ca ^{2 +} concentrations covaried among them and with salinity. The relative abundance of Mg-bearing carbonates, including high-Mg calcite, dolomite and hydrated Ca-magnesite, increased with the salinity. They were absent from the GP subenvironment, where only calcite precipitates, and maximum abundances were found in the GCL subenvironment (61%), where salinity, sulfate, and Mg ²⁺ concentrations were highest. Every subenvironment presented specific microecological characteristics. The microbial community of the GCL subenvironment lacked of oxygenic photosynthesis, while the microbial communities of GML and GP subenvironments were photosynthetically active. Vertical profiles of sulfide and pH at the water-sediment interface revealed clear differences between the GCL and GML subenvironments as well. Sulfide was detected below the oxic layer in the GCL subenvironment and increased with depth, but it was undetected in the GML subenvironment. The precipitation of Mg-bearing carbonates with different Mg:Ca proportions occurs at different stage along a biogeochemical gradient, where increasing salinity and sulfate content favour the anaerobic oxidation of organic carbon by dissimilatory sulfate reduction.     

Cytosolic Nitrate Ion Homeostasis: Could it Have a Role in Sensing Nitrogen Status?

BACKGROUND AND AIMS: question of whether homeostasis occurs for some nutrients and, if so, what are the consequences for how plants sense their nutrient status. Particularly for nitrate, this controversy has focused on the methods used and the cellular pools which they measure. Cytoplasm and cytosol have been distinguished and it has been suggested that two ranges of nitrate values can be separated depending on whether the method separates the pools found in organelles. SCOPE: The present study defines homeostasis of nutrient ions and discusses how whole organ averaging techniques can hide important cellular differences that can help to explain some of the discrepancies between results reported by various methods. These results are considered in relation to a possible role in signalling nutrient status, and have relevance to other averaging techniques such as the use of 'omics' technologies.     

Carbon dioxide transport inxylem causes errors in estimation of rates of respiration in stemsand branches of trees

Complementary laboratory and field experiments showed that theinternal transport of carbon dioxide (CO₂) in the xylemof trees is an important pathway for carbon movement. Carbon dioxidereleased by respiration dissolves in sap and moves upward in thetranspirational stream. The concentration of CO₂ in xylemsap can be up to three orders of magnitude greater than that foundin the atmosphere. In the present experiments, diffusion outwardof a portion of xylem‐transported CO₂ caused a substantialoverestimation of the apparent rate of stem and branch respiration.Rates of CO₂ efflux were linearly related to sap CO₂ concentration.Direct manipulations of xylem sap CO₂ concentration producedrapid and reversible changes in CO₂ efflux from stemsand branches, in some cases quadrupling the rate of efflux. Theseresults demonstrated that apparent rates of stem and branch respirationof trees are in large part a by‐product of the rate of CO₂ diffusionfrom xylem sap.     

Exploring the Radial and Longitudinal Aeration of Primary Maize Roots by Means of Clark-Type Oxygen Microelectrodes

Clark-type oxygen microelectrodes were used to measure the radial and longitudinal oxygen distribution in aerenchymatous and nonaerenchymatous primary roots of intact maize seedlings. A radial intake of oxygen from the rooting medium was restricted by embedding the roots in 1% agar causing aeration to be largely dependent upon longitudinal internal transport from the shoot. In both root types, oxygen concentrations declined with distance from the base, and were lower in the stele than in the cortex. Also, the bulk of the oxygen demand was met internally by transport from the shoots, but a little oxygen was received by radial inward diffusion from the surrounding agar, and in some positions the hypodermal layers received oxygen from both the agar and the cortex. Near to the base, the oxygen partial pressure difference between the cortex and the center of the stele could be as much as 6–8 kPa. Nearer to the tip, the differences were smaller but equally significant. In the nonaerenchymatous roots, cortical oxygen partial pressures near the apex were becoming very low (< 1 kPa) as root lengths approached 100 mm, and towards the center of the stele values reached 0.1 kPa or lower. However, the data indicated that respiratory activity did not decline until the cortical oxygen pressure was less than 2 kPa. Mathematical modeling based on Michaelis–Menten kinetics supported this and suggested that the respiratory decline would be mostly restricted to the stele until cortical oxygen pressures approached very low values. At a cortical oxygen pressure of 0.75 kPa, it was shown that respiratory activity in the pericycle and phloem might remain as high as 80–100% of maximum even though in the center of the stele it could be less than 1% of maximum. Aerenchyma production resulted in increases in oxygen concentration throughout the roots with cortical partial pressures of ca. 5–6 kPa and stelar values of ca. 3–4 kPa near the tips of 100 mm long roots. In aerenchymatous roots, there was some evidence of a decline in the oxygen permeability of the epidermal–hypodermal cylinder close to the apex; a decline in stelar oxygen permeability near the base was indicated for both root types. There was some evidence that the mesocotyl and coleoptile represented a very significant resistance to oxygen transport to the root.     

Submergence-induced leaf acclimation in terrestrial species varying in flooding tolerance

Earlier work on the submergence-tolerant species Rumex palustris revealed that leaf anatomical and morphological changes induced by submergence enhance underwater gas exchange considerably. Here, the hypothesis is tested that these plastic responses are typical properties of submergence-tolerant species. Submergence-induced plasticity in leaf mass area (LMA) and leaf, cell wall and cuticle thickness was investigated in nine plant species differing considerably in tolerance to complete submergence. The functionality of the responses for underwater gas exchange was evaluated by recording oxygen partial pressures inside the petioles when plants were submerged. Acclimation to submergence resulted in a decrease in all leaf parameters, including cuticle thickness, in all species irrespective of flooding tolerance. Consequently, internal oxygen partial pressures (pO(2)) increased significantly in all species until values were close to air saturation. Only in nonacclimated leaves in darkness did intolerant species have a significantly lower pO(2) than tolerant species. These results suggest that submergence-induced leaf plasticity, albeit a prerequisite for underwater survival, does not discriminate tolerant from intolerant species. It is hypothesized that these plastic leaf responses may be induced in all species by several signals present during submergence; for example, low LMA may be a response to low photosynthate concentrations and a thin cuticle may be a response to high relative humidity.