A rapid wavelength changer based on liquid crystal shutters for use in ratiometric microspectrophotometry.

Many of the fluorescent indicator molecules most useful for measuring intracellular concentrations of ions of biological importance, such as Ca2+ or H+, require illumination first at one wavelength, at which the fluorescence depends strongly on the concentration of the ion, and then at another wavelength (e.g. the isosbestic point), so that a ratio can be obtained. Existing wavelength changers are mechanical and involve moving filters, mirrors or gratings. These systems have the disadvantage that they introduce mechanical shocks that can interfere with simultaneous electrophysiological recording. In addition, they require special electrical driving systems and are relatively expensive, especially if they are capable of switching rapidly. We describe a new wavelength changer based on liquid crystal shutters which has the following advantages: (1) it has no mechanical moving parts; (2) it can switch rapidly (@1 ms) and in any desired pattern (off - on1 - off - on2 - off, etc.); (3) it is driven by a low-power 15-V pulse; and (4) it is substantially cheaper than existing wavelength changers. Its limitations are that it does not pass wavelengths shorter than about 400 nm and transmission in the range 430-700 nm is only 20-40%.

Purification of a new dimeric protein from Cliona vastifica sponge, which specifically blocks a non-L-type calcium channel in mouse duodenal myocytes.

Marine sponges are synthesizing a wide variety of peptidic and organic molecules with biological activities. Multiple-step purification of Cliona vastifica extract led to a new dimeric peptide (mapacalcine; M(r) = 19,064) that is composed of two homologous chains, each containing nine cysteins. This protein has been found to selectively block a new calcium conductance characterized in mouse duodenal myocytes with an IC50 value of approximately 0.2 microM. The mapacalcine-sensitive current was a non-L-type calcium current activated from a holding potential of -80 mV that persisted during stimulation of the cell at high frequencies (0.1-0.2 Hz) within 5-10 min. Time constants of inactivation were similar for both L-type and non-L-type calcium currents. The non-L-type calcium current of duodenal myocytes was not blocked by the pharmacological agents specific for N-, L-, P-, or Q-type calcium channels. Mapacalcine was unable to block T-type calcium current in portal vein myocytes as well as voltage-dependent potassium currents and calcium-activated chloride currents in duodenal and portal vein cells. Mapacalcine did not affect caffeine-induced calcium responses, indicating that it did not interfere with intracellular calcium stores. Competition experiments on mouse intestinal membranes showed that mapacalcine did not interact with dihydropyridines receptors. These data suggest that mapacalcine may be a specific inhibitor of a new type of calcium current, first identified in duodenal myocytes.

L-type and Ca2+ release channel-dependent hierarchical Ca2+ signalling in rat portal vein myocytes.

Ca2+ signalling events and whole-cell Ca2+ currents were analyzed in single myocytes from rat portal vein by using a laser scanning confocal microscope combined with the patch-clamp technique. In myocytes in which the intracellular Ca2+ store was depleted or Ca2+ release channels were blocked by 10 microM ryanodine, inward Ca2+ currents induced slow and sustained elevations of [Ca2+]i. These Ca2+ responses were suppressed by 1 microM oxodipine and by depolarizations to +120 mV, a potential close to the reversal potential for Ca2+ ions, suggesting that they reflected Ca2+ influx through L-type Ca2+ channels. With functioning intracellular Ca2+ stores, flash photolysis of caged Ca2+ gave rise to a small increase in [Ca2+]i with superimposed Ca2+ sparks, reflecting the opening of clustered Ca2+ release channels. Brief Ca2+ currents in the voltage range from -30 to +10 mV triggered Ca2+ sparks or macrosparks that did not propagate in the entire line-scan image. Increasing the duration of Ca2+ current for 100 ms or more allowed the trigger of propagating Ca2+ waves which originated from the same initiation sites as the caffeine-activated response. Both Ca2+ sparks and initiation sites of Ca2+ waves activated by Ca2+ currents were observed in the vicinity of areas that excluded the Ca2+ probes, reflecting infoldings of the plasma membrane close to the sarcoplasmic reticulum, as revealed by fluorescent markers. The hierarchy of Ca2+ signalling events, from submicroscopic fundamental events to elementary events (sparks) and propagated waves, provides an integrated mechanism to regulate vascular tone.

In vivo and in vitro studies of the purine-cytosine permease of Saccharomyces cerevisiae. Functional analysis of a mutant with an altered apparent transport constant of uptake.

The FCY2 gene of the purine-cytosine permease (PCP) of Saccharomyces cerevisiae and the allele fcy2-21 have been cloned on the yeast multicopy plasmid pJDB207. The corresponding plasmids were introduced into a S. cerevisiae strain carrying a chromosomal deletion at the FCY2 locus. The resulting strains were designated pAB4 and pAB25 respectively. The pAB25 strain, which carries the fcy2-21 allele, contains four amino acid changes in the open reading frame of the PCP (Weber et al., 1989). The influence of these mutations was studied on cells by determination of the uptake constants of purine bases and cytosine [apparent Michaelis constant of transport (Ktapp) and Vmax] and on plasma-membrane preparations, by measurements of binding parameters at equilibrium [(Kd and maximum amount of binding sites/Bmax)]. For strain pAB4, the Ktapp and Vmax of uptake were almost similar for all solutes considered [1.8-2.6 microM and 8.5-10.2 nmol.min-1.(10(7) cells)-1]. The main effect of the mutations in strain pAB25 was based on a large increase in Ktapp for all ligands except adenine. Plasma membranes of each strain displayed one class of specific binding sites. Variations in Kd of 0.4-1 microM were observed for pAB4. These slight variations had no effect on the Ktapp of uptake measured for the corresponding solutes. In contrast, using pAB25 membranes, Kd increased dramatically; 2.6 microM, 40 microM and 96 microM for adenine, cytosine and hypoxanthine, respectively. These increments were correlated to variations in Ktapp of the uptake for cytosine and hypoxanthine. Therefore, we conclude that modification in the Ktapp of uptake in the strain carrying fcy2-21 allele is merely due to a modification of the binding ability of the permease for its ligands.

Respiration in non-phosphorylating yeast mitochondria. Roles of non-ohmic proton conductance and intrinsic uncoupling.

Respiratory rate, protonmotive force and charge/O ratio were measured under two different kinds of steady state in non-phosphorylating yeast mitochondria: (i) when the electron flux was modulated by a variable limitation in electron supply or (ii) when oxygen consumption was decreased by respiratory chain inhibitor titration. We showed that the relationships between either delta p or charge/O ratio and respiratory rate are different under the two kinds of steady state, indicating different degrees of intrinsic uncoupling in respiratory chain. Moreover, we observed a non-ohmic dependence between H(+)-conductance and delta p. We concluded that the high rate of static-head respiration in yeast mitochondria was determined both by the non-ohmic proton conductance of the inner membrane and the saturation of the redox proton pump slipping.

Calcium-activated chloride current in rat vascular smooth muscle cells in short-term primary culture.

Isolated cells from rat portal vein smooth muscle in short-term primary culture were studied using patch-clamp technique (whole-cell configuration). In order to study a calcium-activated chloride current, the potassium currents were blocked by intracellular cesium diffusion. Without EGTA in the pipette solution, depolarizing voltage pulses from a holding potential of -70 mV to positive potentials activated an early inward and a late outward current. The latter persisted as a long-lasting inward tail current when the membrane was repolarized to -70 mV. The outward current measured at the end of the pulse and the tail current were blocked by extracellular cobalt, after replacement of external calcium with barium, after removal of external calcium, and when the calcium concentration of the pipette solution was less than 0.5 microM, suggesting that they were calcium-dependent. The tail current decay was voltage sensitive, becoming faster with hyperpolarization. The reversal potential of the calcium-activated current was near the equilibrium potential for chloride ions, and was shifted as predicted by the Nernst equation when the extracellular or intracellular chloride concentration was changed. The calcium-activated current was blocked by adding micromolar concentrations of niflumic acid or millimolar concentrations of DIDS. This effect of compounds known to interfere with chloride channels together with the data on the equilibrium potential for chloride ions indicated above suggested the existence of a calcium-activated chloride current in vascular smooth muscle cells.

Comparison of pinaverium bromide, manganese chloride and D600 effects on electrical and mechanical activities in rat uterine smooth muscle.

The effects of pinaverium bromide, were compared with those of D600 and manganese chloride (Mn), on membrane potentials, ionic currents and isometric contractions in uterine smooth muscle strips from pregnant rats. Pinaverium bromide (10(-7) - 10(-6) M) depressed twitch contractions and K-contractures within 15-20 min while D600 (2 X 10(-6) M) and Mn (10(-3) M) abolished both contractions. D600 and pinaverium bromide were more potent inhibitors in K-depolarized preparations than in polarized tissues. At a supramaximal dose (10(-5) M), pinaverium bromide decreased the rate of rise, amplitude, and rate of repolarization of the action potential, and prolonged the potential duration. The inward Ca current was depressed and the reduction in Cai was responsible for the decrease in K current. Pinaverium bromide (10(-5) M) depressed the myometrial contractions induced in Ca-free solution by acetylcholine (10(-4) M) and by prolonged membrane depolarizations. Mn (2.5 X 10(-3) M) only reduced the Ach-induced contraction and D600 (10(-5) M) had no effect on intracellular Ca stores. The results indicate that pinaverium bromide has Ca channel blocking properties similar to those of currently used Ca antagonists; it may also exert an effect to depress contractions supported by intracellular Ca release.