Centimeter-scale stream substratum heterogeneity and metabolic rates

Spatial heterogeneity of substrata in streams may influence dissolved oxygen (O₂) transport and nutrient forms. We studied the relationship between scales of substratum heterogeneity and O₂. Heterogeneous systems could have greater respiration rates as a result of increased interfacial surfaces in the biogeochemically active areas between oxic and anoxic zones. We used grids with twelve 7 × 3.5 cm cells; half the cells were filled with sand and the other half with gravel to quantify the effect of centimeter-scale heterogeneity on respiration. The sand and gravel cells were arranged within the grids to give low, medium, and high heterogeneity. Grids were incubated for 15–17 days in a prairie stream, and then whole grid respiration was analyzed in closed recirculating chambers. Depth to anoxia and substratum metabolism were calculated from O₂ microelectrode profiles measured in each cell of the grid and compared with data from natural stream transects from agricultural, urban, and prairie land use types. Shannon–Weaver (H′) diversity and “probability of change” indices were also used to compare heterogeneity of the grids to the natural stream transects. No significant differences were found among grid heterogeneity levels for respiration rate, but the anoxic interface was deeper in the gravel of higher heterogeneity grids, probably due to greater transport rates of O₂ in the coarse-grained substratum. The H′ and probability of change indices indicated that the grids had levels of heterogeneity within the range of real streams. Grid depth to anoxia and substratum metabolism rates were similar to those found in streams, though less variable. In streams, H′ and probability of change values showed a slight difference among land use types, with some urban and agricultural sites displaying very low heterogeneity. Handling editor: Robert Bailey     

Cyclic AMP and intracellular ionic activities innecturus gallbladder

Summary Open-tip and liquid ion-exchanger microelectrodes were used to study the effects of cAMP (6mm, added to the serosal medium) on apical membrane potential (E _m ) and intracellular sodium, potassium, and chloride activities (a _Na ⁱ ,a _K ⁱ ,a _Cl ⁱ ) inNecturus gallbladder under open-circuit conditions. Transepithelial potential difference (E _Tr ) was also measured. In the presence of cAMP,a _Cl ⁱ fell from about 1.5 times its equilibrium value to a level that corresponded to electrochemical equilibrium across the apical and basolateral cell membranes. Under these conditionsa _Na ⁱ decreased anda _K ⁱ increased,E _m was unchanged andE _Tr increased from virtually zero to a small but significant serosal positive value. The cAMP-induced increase ina _K ⁱ was abolished when Cl^–-free incubation media were used. Addition of the Ca⁺⁺-ionophore A23187 (0.5 g/ml) to the serosal medium had no effect onE _m ,E _Tr , ora _Cl ⁱ . When A23187 was added to the mucosal medium,E _m and the basolateral membrane potential hyperpolarized by about 20 mV and an increase in the outwardly directed electrochemical driving force for Cl^– was observed. These results indicate that cAMP inhibits coupled transapical Na–Cl entry into epithelial cells ofNecturus gallbladder and suggest that this inhibition may not be mediated by an increase in intracellular Ca⁺⁺ concentration.     

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.     

Measurement of intracellular nitrate concentrations in Chara using nitrate-selective microelectrodes

Nitrate-selective microelectrodes have been made using a quaternary ammonium sensor, methyl-tridodecylammonium nitrate, in a Polyvinylchloride matrix. These electrodes showed a log-linear response from 0.1 to 100 mol · m^?3 nitrate with a typical slope of 55.6 mV per decade change in nitrate concentration. The only physiologically significant interfering anion was chloride but the lower limit of nitrate detection was 0.5 mol · m^?3 in the presence of 100 mol · m^?3 chloride which means this interference will not be important in most physiological situations. These microelectrodes were used to measure nitrate concentrations in internodal cells of Chara corallina cultured under low nitrate and nitrate-replete conditions for 6 to 30 weeks. Cells maintained in low nitrate only showed measurements which were less than the detection limit of the electrodes, while cells grown under nitrate-replete conditions showed two populations of measurements having means of 1.6 and 6.2 mol · m^?3. Chemical analysis of the high-nitrate cells indicated that they contained a mean nitrate concentration of 5.9 mol · m^?3. As vacuolar nitrate concentration would dominate this whole-cell measurement, it is concluded that the higher concentration measured with the electrodes represents vacuolar nitrate concentration and the lower value represents the cytoplasmic concentration. This intracellular distribution of nitrate could only be achieved passively if the electrical potential difference across the tonoplast is between +25 and + 35 mV.     

PO2 and pH changes in the retina of the crab Ocypode ryderi: evidence for aerobic glycolysis

Summary The isolated retina of the terrestrial crab Ocypode ryderi exhibits a pronounced lactate production in spite of being supplied with sufficient O₂ (140 torr). To determine whether this lactate production is caused by hypoxic areas in the tissue or represents aerobic glycolysis, oxygen partial pressure and pH measurements with two-channel glass microelectrodes and additional biochemical analyses were carried out on this organ. Distinct profiles were obtained for O₂ partial pressure and pH inside the tissue. At a depth of 200 m different O₂ partial pressure levels could be observed depending on the O₂ partial pressure in the medium (85 torr at 280 torr and 36 torr at 130 torr, respectively). The extracellular pH displays a similar pattern; it reaches a stable value of 7.15 at 100 m inside the tissue. Lowering bath O₂ partial pressure from 280 torr to about 15 torr (hypoxia) induces a decrease of the O₂ partial pressure in the tissue with different time-courses for different tissue depths. However, hypoxia did not change the extracellular pH. Addition of antimycin A (100 mol · 1^-1) to the medium abolishes the O₂ partial pressure gradient and the delayed recovery of the tissue O₂ partial pressure after hypoxia. These results and the biochemical data suggest that in the crab retina a high glycolytic activity occurs simultaneously with oxydative carbohydrate degradation (aerobic glycolysis).Abbreviations AEC Atkinson energy charge - DC bioelectric potential - dw dry weight - HEPES N-[2-Hydroxyethyl]piperazine-N-[2-ethanesulphonic acid] - PCO₂ carbon dioxide partial pressure - PO₂ oxygen partial pressure - P _tO₂ oxygen partial pressure inside the tissue - P _mO₂ oxygen partial pressure in the medium - pH_t pH inside the tissue - pH_m pH in the superfusion medium     

pH-stat experiments in proximal renal tubules

Summary The pH-stat technique has been used to measure H⁺ fluxes in gastric mucosa and urinary bladder in vitro while keeping mucosal pH constant. We now report application of this method in renal tubules. We perfused proximal tubules with double-barreled micropipettes, blocked luminal fluid columns with oil and used a double-barreled Sb/reference microelectrode to measure pH, and Sb or 1n HC1-filled microelectrodes to inject OH^– or H⁺ ions into the tubule lumen. By varying current injection, pH was kept constant at adjustable levels by an electronic clamping circuit. We could thus obtain ratios of current (nA) to pH change (apparent H⁺-ion conductance). These ratios were reduced after luminal 10^–4 m acetazolamide, during injection of OH^–, but they increased during injection of H⁺. The point-like injection source causes pH to fall off with distance from the injecting electrode tip even in oil-blocked segments. Therefore, a method analogous to cable analysis was used to obtain H⁺ fluxes per cm² epithelium. The relation betweenJ _H ⁺ and pH gradient showed saturation kinetics of H fluxes, both during OH^– and H⁺ injection. This kinetic behavior is compatible with inhibition ofJ _H by luminal H⁺. It is also compatible with dependence on Na⁺ and H⁺ gradients of a saturable Na/H exchanger. H⁺-ion back-flux into the tubule lumen also showed saturation kinetics. This suggests that H⁺ flow is mediated by a membrane component, most likely the Na⁺–H⁺ exchanger.     

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.     

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.