The kinetics of calcium and magnesium entry into mycorrhizal spruce roots.

The entry of calcium and magnesium from external sources into mycorrhizal roots of 3-year-old Norway spruce trees (Picea abies [L.] Karst.) was monitored. Roots of intact plants were exposed for various periods of time, ranging from 2 min to 48 h, to nutrient solutions which contained the stable-isotope tracers 25Mg and 44Ca. After labelling, samples of roots were excised from the plants, shock-frozen, cryosubstituted and embedded. The resulting isotope composition in this material was analysed by a laser-microprobe-mass-analyser (LAMMA) at relevant positions within cross-sections of the roots. For both elements, we determined (i) the fractions of the isotopes originating from the plant prior to labelling, and (ii) the fraction of isotopes originating from the corresponding tracer that penetrated into the root. Both divalent cations rapidly penetrated across the cortical apoplast and reached the endodermis. After 2 min of exposure to the labelling solution, an initial transient gradient of the tracers could be observed within the root cortex. Subsequently, calcium as well as magnesium equilibrated between the apoplast of the entire cortex and the external tracer with a half-time, t1/2, of about 3 min. In contrast, the kinetics of radial movement into the vascular stele showed a delay with a t1/2 of 100-120 min. We take this as strong evidence that there exists a free apoplastic path for divalent cations in the cortex and that the endodermis is a major barrier to the further passage of Mg and Ca into the xylem. While 25Mg in the labelling solution exchanged rapidly with Mg in the cortical apoplast, the exchange across the plasma membrane with Mg present in the protoplasm of the same cortical cells was almost 2 orders of magnitude slower. The kinetics of Ca and Mg entry at + 6 degrees C were similar to those obtained at a root temperature of +22 degrees C.

A depolarizing chloride current contributes to chemoelectrical transduction in olfactory sensory neurons in situ.

Recent biophysical investigations of vertebrate olfactory signal transduction have revealed that Ca2+-gated Cl- channels are activated during odorant detection in the chemosensory membrane of olfactory sensory neurons (OSNs). To understand the role of these channels in chemoelectrical signal transduction, it is necessary to know the Cl--equilibrium potential that determines direction and size of Cl- fluxes across the chemosensory membrane. We have measured Cl-, Na+, and K+ concentrations in ultrathin cryosections of rat olfactory epithelium, as well as relative element contents in isolated microsamples of olfactory mucus, using energy-dispersive x-ray microanalysis. Determination of the Cl- concentrations in dendritic knobs and olfactory mucus yielded an estimate of the Cl--equilibrium potential ECl in situ. With Cl- concentrations of 69 mM in dendritic knobs and 55 mM in olfactory mucus, we obtained an ECl value of +6 +/- 12 mV. This indicates that Ca2+-gated Cl- channels in olfactory cilia conduct inward currents in vivo carried by Cl- efflux into the mucus. Our results show that rat OSNs are among the few known types of neurons that maintain an elevated level of cytosolic Cl-. In these cells, activation of Cl- channels leads to depolarization of the membrane voltage and can induce electrical excitation. The depolarizing Cl- current in mammalian OSNs appears to contribute a major fraction to the receptor current and may sustain olfactory function in sweet-water animals.

Accumulation of calcium in degenerating photoreceptors of several Drosophila mutants.

The hypothesis that a large, possibly toxic, increase in cellular calcium accompanies photoreceptor cell degeneration in several different Drosophila mutants was tested. The calcium content of wild type and mutant photoreceptors of Drosophila was measured using rapid freezing of the eyes and energy-dispersive x-ray analysis (e.d.x.) of cryosections and semithin sections of cryosubstituted material. Light- and dark-raised mutants of the following strains were studied: retinal degeneration B (rdgB); retinal degeneration C (rdgC); neither inactivation nor afterpotential C (ninaC), and no receptor potential A (norpA). These are light-dependent retinal degeneration mutants in which the affected gene products had been previously shown as myosin-kinase (ninaC), calcium-dependent phosphoprotein phosphatase (rdgC), phosphoinositide transfer protein (rdgB), and phospholipase C (norpA). In light-raised mutants, ommatidia of variable degrees of degeneration were observed. Mass-dense globular bodies of 200-500 nm diameter in relatively large quantities were found in the degenerating photoreceptor of all the mutants tested. These subcellular globules were found to have a very high calcium content, which was not found in wild type or in nondegenerating photoreceptors of the mutants. Nondegenerating photoreceptors were found not only in dark-raised mutants, but in smaller quantities also in light-raised mutants. Usually these globular structures contained high levels of phosphorus, indicating that at least part of the calcium in the mutant photoreceptors is precipitated as calcium phosphate. The results indicate that a large increase in cellular calcium accompanies light-induced photoreceptor degeneration in degenerating Drosophila mutants even when induced by very different mutations, suggesting that the calcium accumulation is a secondary rather than a primary effect in the degeneration process.

Light-induced calcium release and re-uptake in toad rods.

Laser micromass analysis was used to investigate the effect of light on the Ca content of rod outer segments in the isolated retina and eyecup of the toad, Bufo marinus. Isolated retinas were incubated in Ringer for which most of the Ca (normally 97% 40Ca) was replaced with 44Ca, so that release of internal Ca (as 40Ca) could be distinguished from the uptake of 44Ca from the external medium. Continuous illumination produced a decline in outer segment 40Ca content. In bright light, the decrease in 40Ca was sigmoidal, beginning with a delay of at least 4 sec, reaching a maximal rate of 1-2 x 10(8) Ca/rod/sec after 8-16 sec, and declining to zero as the light-releasable pool of 40Ca was exhausted within 1-2 min. Decreases of similar magnitude and time course to those observed in isolated retina were also seen in an eyecup preparation. The rate of light-dependent release was reduced 20- to 60-fold by substitution of external Na with choline or Li. The rate of 44Ca uptake from the external medium into rods was little affected during continuous illumination. Uptake, however, was markedly increased in darkness following light exposure. This Ca re-uptake occurred at a rate approximately 2-fold greater in eyecups than in isolated retinas. We interpret our results to show that light causes an increase in the permeability or transport of Ca across the disk membrane and that the Ca is extruded from the rod via Na/Ca (or Na/Ca-K) exchange across the plasma membrane. Cessation of illumination stimulates the resequestration of Ca back into the disks, and this process is somehow enhanced by the presence of the pigment epithelium.

Calcium in dark-adapted toad rods: evidence for pooling and cyclic-guanosine-3'-5'-monophosphate-dependent release.

1. We have used laser micromass analysis (l.a.m.m.a.) to investigate Ca uptake and release in intact 'red' rod photoreceptors in the dark-adapted retina of the toad, Bufo marinus. 2. With l.a.m.m.a. it is possible to measure separately the concentrations of each of the Ca isotopes. Rods normally containing almost exclusively 40Ca can be incubated in Ringer solution containing the stable isotopes 42Ca or 44Ca. In this way, the movements of Ca into and out of the rod can be separately determined. 3. When rods are incubated in darkness in high 44Ca (up to 20 mM), large amounts of 44Ca accumulate in the outer segment at a rate which increases with increasing external 44Ca concentration. However, this 44Ca appears not to exchange with the 40Ca originally present within the rod. This result suggests that the 40Ca may be sequestered within a pool which normally exchanges slowly with external Ca. 4. We explored Ca exchange in high-Ca solutions in more detail with double-isotope labelling. In these experiments, rods were first pre-loaded with Ca of one isotope (42Ca) and then incubated in Ringer solution containing another (44Ca). We could then measure separately the rate of exchange of the pre-loaded 42Ca with the 44Ca in the Ringer solution and with the 40Ca originally present within the rod in the sequestered pool. 5. These experiments show that the pre-loaded-Ca exchanges rapidly with Ca in the Ringer solution, at least in part by Ca-Ca exchange, but much more slowly with the Ca originally present within the rod. Thus Ca in the outer segments can exist in (at least) two pools: one which exchanges rapidly across the plasma membrane and is probably Ca free or loosely bound within the cytosol, and another which exchanges slowly and is probably Ca within the disks. 6. Although Ca sequestered within the outer segment normally exchanges quite slowly, it can be rapidly released if the extracellular free Ca is buffered to low levels with EGTA. The rate-limiting step for Ca release under these conditions appears not to be Na-Ca exchange, since the rate of Ca efflux is unchanged if the Na in the Ringer solution is substituted with choline. 7. Ca can also be released from the sequestered pool if rods are incubated in Ringer solution containing 100 or 500 microM-IBMX (3-isobutyl-1-methylxanthine).(ABSTRACT TRUNCATED AT 400 WORDS)

Calcium content and calcium exchange in dark-adapted toad rods.

We have used laser-activated micro mass analysis (l.a.m.m.a.) and energy-dispersive X-ray analysis (e.d.x.) to measure Ca content and Ca movements in 'red' rod photoreceptors in the dark-adapted retina of the toad, Bufo marinus. Measurements with both l.a.m.m.a. and e.d.x. show that intact rod outer segments contain 4-5 mmol total Ca/l wet tissue volume, or 1-2 Ca per rhodopsin. We could detect no significant variation in the total Ca as a function of distance across or up and down the outer segment. In the inner segment, Ca could be detected only within the mitochondria-rich ellipsoid body, where the total Ca concentration was of the order of 100-400 mumol/l wet tissue volume. To measure the exchange of Ca in outer segments from intact photoreceptors, we exposed the dark-adapted retina to Ringer containing the stable isotope 44Ca. Since l.a.m.m.a. can measure separately the concentrations of each of the isotopes of the elements, and since native rods contain almost exclusively 40Ca, the increase in 44Ca and decrease in 40Ca could be used as a measure of Ca influx and efflux. Ca exchange in intact rod outer segments in darkness is very slow. The rate of accumulation of 44Ca was only 10(5) Ca/rod.s, or about 10% of the total outer segment Ca/h. This slow rate of exchange is apparently not the result of restricted movement of Ca across the plasma membrane. Ca exchange was also measured in outer segments which were either partially or entirely detached from the rest of the photoreceptor. In broken-off outer segments, Ca exchange is faster than in the intact organelles, and in 1 h, half of the 44Ca exchanges for 40Ca. When the retina was incubated in Ringer for which all of the Na was substituted with Li or choline, there was an increase in the rate of 44Ca accumulation in intact outer segments, probably due to an inhibition of Na-Ca counter transport across the plasma membrane. Our measurements indicate that the great majority of the Ca in the rod appears to be inaccessible to exchange under physiological conditions, probably because it is sequestered within the disks which in intact rods appear to be nearly impermeable to Ca in darkness.

Light-dependent calcium release from photoreceptors measured by laser micro-mass analysis.

Yoshikami and Hagins first suggested that calcium is sequestered within membranous disks in the outer segments of vertebrate rods and that the bleaching of visual pigment molecules by light causes the release of Ca from the disks. Once released, the Ca was postulated to bind to Na+ channels or carrier molecules in the plasma membrane to produce the electrical response. This theory, termed the 'calcium hypothesis', is supported by much evidence but remains controversial, largely because of the difficulty in measuring calcium in rods and of demonstrating light-induced release. Here we describe direct measurements of total rod Ca using a new microprobe method, called laser micro-mass analysis, or LAMMA . Using this technique, we show that rods contain large amounts of Ca concentrated in their outer segments. Physiological levels of illumination produce a graded efflux of rod Ca content, amounting to about 10(4) ions per rhodopsin molecule bleached in dim light. As light does not change the rate of Ca influx, the total Ca content of the rod decreases. In bright light, as much as half the total Ca leaves the rod during only 1 min of illumination.