Effects of caffeine and raynodine on low pHi-induced changes in gap junction conductance and calcium concentration in crayfish septate axons

Summary Electrical uncoupling of crayfish septate axons with acidification has been shown to cause a substantial increase in [Ca²⁺]_i which closely matches in percent the increase in junctional resistance. To determine the origin of [Ca²⁺]_i increase, septate axons have been exposed either to drugs that influence Ca²⁺ release from internal stores, caffeine and ryanodine, or to treatments that affect Ca²⁺ entry. A large increase in junctional resistance and [Ca²⁺]_i maxima above controls resulted from addition of caffeine (10–30mm) to acetate solutions, while a substantial decrease in both parameters was observed when exposure to acetate-caffeine was preceded by caffeine pretreatment. In contrast, ryanodine (1–10 m) always caused a significant decrease in junctional resistance and [Ca²⁺]_i maxima when applied either together with acetate or both before and with acetate. Calcium channel blockers such as La³⁺, Cd²⁺ and nisoldipine had no effect, while an increase in the [Ca²⁺] of acetate solutions either decreased junctional resistance and [Ca²⁺]_i maxima or had no effect. The data suggest that cytoplasmic acidification causes an increase in [Ca²⁺]_i by releasing Ca²⁺ from caffeine and ryanodine-sensitive Ca²⁺ stores. The increase in [Ca²⁺]_i results in a decrease in gap junction conductance.     

Ca modulates outward current throughI K1 channels

Summary Inward-rectifier channels in cardiac cells (I _K1) stabilize the resting membrane potential near the K equilibrium potential. Here we investigate the role ofI _K1 in the regulation of action potentials and link this to the influx of Ca during beating. Inward Ca current alters the open-channel probability of outwardI _K1 current. Thus Ca ions depolarize cells not only by carrying an inward current but also by blocking an outward current.     

A nonselective cation channel activated by membrane deformation in oocytes of the ascidianBoltenia villosa

Summary Cell-attached patch clamp recordings from unfertilized oocytes of the ascidianBoltenia villosa reveal an ion channel which is activated by mechanical deformation of the membrane. These channels are seen when suction is applied to the patch pipette, but not in the absence of suction or during voltage steps. The estimated density of these stretch-activated channels is about 1.5/m², a figure equal to or greater than the density of known voltage-dependent channels in the oocyte. Ion substitution experiments done with combined whole-cell and attached patch recording, so absolute potentials are known, indicate that the channel passes Na⁺, Ca²⁺ and K⁺, but not Cl^–. The channel has at least two open and two closed states, with the rate constant that leaves the longer-lived closed state being the primary site of stretch sensitivity. External Ca²⁺ concentration affects channel kinetics: at low calcium levels, long openings predominate, whereas at high calcium virtually all openings are to the short-lived open state. In multiple channel patches, the response to a step change in suction is highly phasic, with channel open probability decreasing over several hundred milliseconds to a nonzero steady-state level after an initial rapid increase. This channel may play a role in the physiological response of cells of the early embryo to the membrane strains associated with morphogenetic events.     

Activation and conductance properties of ryanodine-sensitive calcium channels from brain microsomal membranes incorporated into planar lipid bilayers

Summary Rat brain microsomal membranes were found to contain high-affinity binding sites for the alkaloid ryanodine (k _d 3nm.B _max 0.6 pmol per mg protein). Exposure of planar lipid bilayers to microsomal membrane vesicles resulted in the incorporation, apparently by bilayer-vesicle fusion, of at least two types of ion channel. These were selective for Cl^– and Ca²⁺, respectively. The reconstituted Ca²⁺ channels were functionally modified by 1 m ryanodine, which induced a nearly permanently open subconductance state. Unmodified Ca²⁺ channels had a slope conductance of almost 100 pS in 54mm CaHEPES and a Ca²⁺/TRIS⁺ permeability ratio of 11.0. They also conducted other divalent cations (Ba²⁺>Ca²⁺>Sr²⁺>Mg²⁺) and were markedly activated by ATP and its nonhydrolysable derivative AMPPCP (1mm). Inositol 1,4,5-trisphosphate (1–10 m) partially activated the same channels by increasing their opening rate. Brain microsomes therefore contain ryanodine-sensitive Ca²⁺ channels, sharing some of the characteristics of Ca²⁺ channels from striated but not smooth muscle sarcoplasmic reticulum. Evidence is presented to suggest they were incorporated into bilayers following the fusion of endoplasmic reticulum membrane vesicles, and their sensitivity to inositol trisphosphate may be consistent with a role in Ca²⁺ release from internal membrane stores.     

Barium- and calcium-permeable channels open at negative membrane potentials in rat ventricular myocytes

Summary Ca²⁺- and Ba²⁺-permeable channel activity from adult rat ventricular myocytes, spontaneously appeared in the three single-channel recording configurations: cell-attached, and excised inside-out or outside-out membrane patches. Single-channel activity was recorded at steady-state applied membrane potentials including the entire range of physiologic values, and displayed no rundown in excised patches. This activity occurred in irregular bursts separated by quiescent periods of 5 to 20 min in cell-attached membrane patches, whereas in excised patch experiments, this period was reduced to 2 to 10 min. During activity, a variety of kinetic behaviors could be observed with more or less complex gating patterns. Three conductance levels: 22, 45 and 78 pS were routinely observed in the same excised membrane patch, sometimes combining to give a larger level. These channels were significantly permeable to divalent cations and showed little or no permeability to potassium or sodium ions. The inorganic blockers of voltage-gated Ca channels, cobalt (2mm), cadmium (0.5mm) or nickel (3mm), had no apparent effect on these spontaneous unitary currents carried by barium ions. Under 10^–5 m bay K 8644 or nitrendipine, the activity was clearly increased in about half of the tested excised inside-out membrane patches. Both dihydropyridines enhanced openings of the larger conductance level, which was only very occasionally seen under control conditions. When the single-channel activity became sustained under 5×10^–6 m Bay K 8644, it was possible to calculate the mean unitary current at different membrane potentials and show that the mean current value increased with membrane potential.     

Aluminium interactions with K+(86Rb+) and 45Ca2+ fluxes in three cultivars of sugar beet (Beta vulgaris)

Three cultivars of sugar beet (Beta vulgaris L.), which are sensitive to aluminium (Al) in the order Primahill > Monohill > Regina, were grown in water culture for 2 weeks. Nutrients were supplied at 15% increase of amounts daily, corresponding to the nutrient demand for maximal growth. The 2.4-dinitrophenol (DNP)-sensitive (metabolic) and DNP-insensitive (non-metabolic) uptake of aluminium, phosphate. ⁴⁵Ca²⁺ and K⁺(⁸⁶Rb⁺) in roots were measured as well as transport to shoots of intact plants. All 3 cultivars absorbed more aluminium if DNP was present during the aluminium treatment than in its absence. It is suggested that sugar beets are able to extrude aluminium activity or that they possess an active mechanism to keep Al outside the cell. The presence of Al in the medium during the 1-h experiment affected the metabolic and non-metabolic fluxes of ⁴⁵Ca²⁺ and K⁺(⁸⁶Rb⁺) in different ways. In the presence of DNP, the influx of both ⁴⁵Ca²⁺ and K⁺(⁸⁶Rb⁺) and the efflux of ⁴⁵Ca²⁺ were inhibited by Al in a competitive way. At inhibition of ⁴⁵Ca²⁺ influx, 2 Al ions are probably bound per Ca²⁺ uptake site in cv. Regina (Al-tolerant), but in cvs Primahill and Monohill only one Al ion is bound (more Al sensitive). Aluminium competitively inhibited the active efflux of ⁴⁵Ca²⁺ (absence of DNP) in almost the same way in the 3 cultivars. In contrast, aluminium stimulated the influx of K⁺(⁸⁶Rb⁺) in cvs Primahill, Monohill and Regina in the absence of DNP. Thus, the Al effects on active and passive K⁺(⁸⁶Rb⁺) influx are different. The total influx of K⁺(⁸⁶Rb⁺) increased in the presence of Al and might be connected to an active exclusion of Al. Regina is the least Al-sensitive cultivar, probably because Al interferes less with the Ca²⁺ fluxes and because this cultivar actively excludes phosphate in the presence of Al. Thus Al-phosphate precipitation within the plant could be avoided.     

Effect of Ca on composition of fat body of peanut seed

Peanut fruits were grown in nutrient media with or without Ca and in a soil with two Ca levels, from the 20th day after penetration of the gynophore. Seed weight was smaller in the nutrient medium without Ca than in the nutrient medium with Ca, and it was also smaller in the soil with 4 meq of exchangeable Ca (L treatment) than in the soil with 10 meq of exchangeable Ca (H treatment). The fat body of seeds from the Ca deficient medium and the L treatment had a decreased phospholipid content and an increased simple lipid content. In the seed from the H treatment, phosphatidylcholine increased from the 30th to 60th day, while caldiolipin decreased. The amount of triglyceride in the simple lipid content of fat body was decreased by Ca deficiency in the nutrient medium, whereas that of diglyceride was increased, but these effects were not observed in the fat body of the seed from the L treatment. No effect of Ca deficiency was observed in the fatty acid composition of triglyceride.     

Growth responses of rice in ammonium-based nutrient solution with variable calcium supply

It is commonly known that calcium promotes NO₃ ^- uptake in many crop species. However, calcium enhancement of NH₄ ⁺ uptake by plants has received little attention. This study aimed to evaluate the effect of Ca supplements on NH₄ ⁺ uptake and plant growth in solution cultured rice. Supplemental Ca applied at vegetative and reproductive phases of plant ontogeny tended to stimulate NH₄ ⁺ absorption, and accordingly resulted in a better straw and grain yield. However, excessively supplied Ca (400 ppm) was detrimental to plant growth. Increases in straw and grain yield observed at Ca levels up to 300 ppm were linked to the Ca-enhanced activities of glutamine synthetase (GS), glutamate synthase (GOGAT), and ribulose 1, 5-bisphosphate carboxylase/oxygenase (Rubisco).     

Oxidation of NADH by plant mitochondria: Kinetics and effects of calcium ions

The kinetics of NADH oxidation by the outer membrane electron transport system of intact beetroot (Beta vulgaris L.) mitochondria were investigated. Very different values for V_max and the K_m for NADH were obtained when either antimycin A-insensitive NADH-cytochrome c activity (V_max= 31 ± 2.5 nmol cytochrome c (mg protein)^?1 min^?1; K_m= 3.1 ± 0.8 μM) or antimycin A-insensitive NADH-ferricyanide activity (V_max= 1.7 ± 0.7 μmol ferricyanide (mg protein)^?1 min^?1; K_m= 83 ± 20 μM) were measured. As ferricyanide is believed to accept electrons closer to the NADH binding site than cytochrome c, it was concluded that 83 ± 20 μM NADH represented a more accurate estimate of the binding affinity of the outer membrane dehydrogenase for NADH. The low K_m determined with NADH-cytochrome c activity may be due to a limitation in electron flow through the components of the outer membrane electron transport chain. The K_m for NADH of the externally-facing inner membrane NADH dehydrogenase of pea leaf (Pisum sativum L. cv. Massey Gem) mitochondria was 26.7 ± 4.3 μM when oxygen was the electron acceptor. At an NADH concentration at which the inner membrane dehydrogenase should predominate, the Ca²⁺ chelator, ethyleneglycol-(β-aminoethylether)-N,N,-tetraacetic acid (EGTA), inhibited the oxidation of NADH through to oxygen and to the ubiquinone-10 analogues, duroquinone and ubiquinone-1, but had no effect on the antimycin A-insensitive ferricyanide reduction. It is concluded that the site of action of Ca²⁺ involves the interaction of the enzyme with ubiquinone and not with NADH.