Bone as an ion exchange organ: evidence for instantaneous cell-dependent calcium efflux from bone not due to resorption.

The current study tests the hypothesis that basal level and minute-by-minute correction of plasma Ca2+ by outward and inward Ca2+ fluxes from and into an exchangeable ionic pool in bone is controlled by an active partition system without contributions from the bone remodeling system. Direct real-time measurements of Ca2+ fluxes were made using the scanning ion-selective electrode technique (SIET) on living bones maintained ex vivo in physiological conditions. SIET three-dimensional measurements of the local Ca2+ concentration gradient (10 microm spatial resolution) were performed on metatarsal bones of weanling mice after drilling a 100-mum hole through the cortex to expose the internal bone extracellular fluid (BECF) to the bathing solution, whose composition mimicked the extracellular fluid (ECF). Influxes of Ca2+ towards the center of the cortical hole (15.1+/-4.2 pmol cm-2 s-1) were found in the ECF and were reversed to effluxes (7.4+/-2.9 pmol cm-2 s-1) when calcium was depleted from the ECF, mimicking a plasma demand. The reversal from influx to efflux and vice versa was immediate and fluxes in both directions were steady throughout the experimental time (>or=2 h, n=14). Only the efflux was nullified within 10 min by the addition of 10 mM/L Na-Cyanide (n=7), demonstrating its cell dependence. The timeframes of the exchanges and the stability of the Ca2+ fluxes over time suggest the existence of an exchangeable calcium pool in bone. The calcium efflux dependency on viable cells suggests that an active partition system might play a central role in the short-term error correction of plasma calcium without the contribution of bone remodeling.

Covariance of ion flux measurements allows new interpretation of Xenopus laevis oocyte physiology.

An animal-vegetal net ionic current identified previously using voltage probe techniques in maturing Xenopus laevis oocytes has now been investigated using noninvasive ion-selective microelectrodes. Three-dimensional fluxes of hydrogen (H(+)), potassium (K(+)), and bicarbonate (HCO(3)(-)) were characterized with respect to the developmental stage and hemisphere of the oocyte and presence of surrounding follicular tissue. Variable effluxes of H(+) and HCO(3)(-) were recorded from both the animal and vegetal hemispheres. Variable influxes and effluxes of K(+) were also observed. The equatorial region, silent by voltage probe, exhibited fluxes of H(+) and K(+). Simultaneous measurement of pairs of ions allowed correlation analysis of two ion types. Notably for H(+) and K(+) data, positive and negative correlation at animal and vegetal poles respectively offer an explanation of the unpredictable results obtained when individual ions were observed independently.

Cellular oscillations and the regulation of growth: the pollen tube paradigm.

The occurrence of oscillatory behaviours in living cells can be viewed as a visible consequence of stable, regulatory homeostatic cycles. Therefore, they may be used as experimental windows on the underlying physiological mechanisms. Recent studies show that growing pollen tubes are an excellent biological model for these purposes. They unite experimental simplicity with clear oscillatory patterns of both structural and temporal features, most being measurable during real-time in live cells. There is evidence that these cellular oscillators involve an integrated input of plasma membrane ion fluxes, and a cytosolic choreography of protons, calcium and, most likely, potassium and chloride. In turn, these can create positive feedback regulation loops that are able to generate and self-sustain a number of spatial and temporal patterns. Other features, including cell wall assembly and rheology, turgor, and the cytoskeleton, play important roles and are targets or modulators of ion dynamics. Many of these features have similarities with other cell types, notably with apical-growing cells. Pollen tubes may thus serve as a powerful model for exploring the basis of cell growth and morphogenesis. BioEssays 23:86-94, 2001.

Developmental fate of the yolk protein lipovitellin in embryos and larvae of winter flounder, Pleuronectes americanus.

The developmental fate of the vitellogenin-derived yolk protein, lipovitellin (Lv), was investigated in winter flounder embryos and yolk-sac larvae. Since Lv is present as only one major polypeptide in ovulated winter flounder eggs, unlike the multiple yolk polypeptides found in the mature eggs of most teleosts, this system is presented as a simpler model of yolk protein structure and utilization during teleostean development. Winter flounder Lv is cleaved during embryogenesis from a 94 kD polypeptide at fertilization to 67 kD and 26 kD polypeptides at hatching. The rate of this proteolytic processing is slow during early embryonic development, but enters a more rapid phase between days 8 and 12 post-fertilization in embryos reared at 4-5 degrees C, and approaches 50% completion at day 10. Lv processing is essentially complete 3 days before hatching; nevertheless, major degradation of the Lv peptide by the developing winter flounder does not occur until after hatching. The Stokes radius of Lv changes only moderately following processing, from 4.50 nm in unfertilized eggs to 4.19 nm in late embryos and newly hatched larvae, whereas the processed Lv retains its heat stability relative to other yolk polypeptides. Nearly 50% of its lipid content, however, is released from the Lv particle during embryogenesis, concomitant with cleavage of the Lv 94 kD polypeptide. Lv processing may thus render a portion of the yolk protein-associated lipid more accessible to the developing embryo, whereas other yolk components are retained for later use by the winter flounder larva. Alternately, removal of lipid may lead to proteolytic vulnerability of the Lv polypeptide. In either case, only a portion of the lipid moiety of the Lv particle appears to play a significant nutritive role for the embryo, whereas its protein component is reserved for larval use. J. Exp. Zool. 284:686-695, 1999.

Growing pollen tubes possess a constitutive alkaline band in the clear zone and a growth-dependent acidic tip.

Using both the proton selective vibrating electrode to probe the extracellular currents and ratiometric wide-field fluorescence microscopy with the indicator 2', 7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF)-dextran to image the intracellular pH, we have examined the distribution and activity of protons (H+) associated with pollen tube growth. The intracellular images reveal that lily pollen tubes possess a constitutive alkaline band at the base of the clear zone and an acidic domain at the extreme apex. The extracellular observations, in close agreement, show a proton influx at the extreme apex of the pollen tube and an efflux in the region that corresponds to the position of the alkaline band. The ability to detect the intracellular pH gradient is strongly dependent on the concentration of exogenous buffers in the cytoplasm. Thus, even the indicator dye, if introduced at levels estimated to be of 1.0 microM or greater, will dissipate the gradient, possibly through shuttle buffering. The apical acidic domain correlates closely with the process of growth, and thus may play a direct role, possibly in facilitating vesicle movement and exocytosis. The alkaline band correlates with the position of the reverse fountain streaming at the base of the clear zone, and may participate in the regulation of actin filament formation through the modulation of pH-sensitive actin binding proteins. These studies not only demonstrate that proton gradients exist, but that they may be intimately associated with polarized pollen tube growth.

Characterization of a heat-stable fraction of lipovitellin and development of an immunoassay for vitellogenin and yolk protein in winter flounder (Pleuronectes americanus).

An enzyme-linked immunoabsorbent assay was developed for detection and quantification of the yolk protein lipovitellin (Lv) and its plasma precursor, vitellogenin (Vg), in winter flounder (Pleuronectes americanus). Native Lv was found to be a mixture of heat-stable and heat-labile molecules in mature, ovulated eggs. A heat-stable Lv fraction was purified from extracts of unfertilized eggs by brief heat treatment and gel permeation chromatography on Bio-Gel A-1.5. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of heat-stable Lv revealed a single polypeptide of 94 kD, while native Lv also possessed several smaller polypeptides, suggesting that heat-labile Lv contains proteolytic cleavages of the 94-kD polypeptide which destabilize its structure. The Stokes radius of the native protein on Bio-Gel A-1.5 was estimated at 4.50 nm, while the Stokes radii of heat-stable and heat-labile Lv were 4.26 nm and 5.17 nm, respectively. Heat-stable Lv was used to produce a rabbit polyclonal antiserum which reacted with a single 175-kD polypeptide in Western blots of vitellogenic female winter flounder serum, but did not react with any component of male serum. Ouchterlony double diffusion using this antiserum demonstrated immunological identity of Lv, heat-stable Lv, and Vg. The anti-Lv anti-serum was used to construct an homologous ELISA with a linear response between 25 and 300 ng/ml. This assay was used to characterize a Bio-Gel A-1.5 column profile of serum from an estradiol-treated male winter flounder, and a single peak, with Stokes radius of 6.70 nm, was identified as Vg. Winter flounder Vg was confirmed to be a dimer, while Lv from mature eggs was found to be a monomer of a lower molecular weight polypeptide.

Pollen Tube Growth and the Intracellular Cytosolic Calcium Gradient Oscillate in Phase while Extracellular Calcium Influx Is Delayed.

Ratio images of cytosolic Ca2+ (Ca2+i) in growing, fura-2-dextran-loaded Lilium longiflorum pollen tubes taken at 3- to 5-sec intervals showed that the tip-focused [Ca2+]i gradient oscillates with the same period as growth. Similarly, measurement of the extracellular inward current, using a noninvasive ion-selective vibrating probe, indicated that the tip-directed extracellular Ca2+ (Ca2+o) current also oscillates with the same period as growth. Cross-correlation analysis revealed that whereas the [Ca2+]i gradient oscillates in phase with growth, the influx of Ca2+o lags by ~11 sec. Ion influx thus appears to follow growth, with the effect that the rate of growth at a given point determines the magnitude of the ion influx ~11 sec later. To explain the phase delay in the extracellular inward current, there must be a storage of Ca2+ for which we consider two possibilities: either the inward current represents the refilling of intracellular stores (capacitative calcium entry), or it represents the binding of the ion within the cell wall domain.

Effects of maternal nutrition and egg provisioning on parameters of larval hatch, survival and dispersal in the gypsy moth, Lymantria dispar L.

North American gypsy moths disperse as newly hatched larvae on wind currents in a behavior called ballooning. Because ballooning occurs before neonates begin to feed, resources used in dispersal are limited to those carried over from the egg. We show that nutritional experience of the maternal parent can influence the tendency of offspring to disperse, and that resource provisioning of eggs by the maternal parent affects the duration of the window for disperal. Offspring of females from defoliated sites had a lower tendency to balloon in a wind tunnel than larvae from females which had not experienced nutritional stress associated with host defoliation. The number of eggs in an egg mass, a reflection of the maternal parent's nutritional experience, also contributed to the predictive model for dispersal that included defoliation level. Egg weight and the levels of two yolk proteins, vitellin (Vt) and glycine-rich protein (GRP), however, had no influence of the proportion of ballooning larvae. The length of survival without food, and thus the maximum period of time for dispersal, was correlated with levels of Vt and GRP, but not with egg weight. The level of defoliation at the site from which the maternal parent was collected was not related to the longevity of offspring, nor did it have a significant effect on the levels of Vt, GRP or egg weight. Levels of hemolymph proteins arylphorin and vitellogenin in the maternal parent during the prepupal stage had no influence on levels of yolk proteins, larval longevity, or tendency to balloon.

Follicle cell calmodulin in Blattella germanica: transcript accumulation during vitellogenesis is regulated by juvenile hormone.

There is abundant calmodulin (CaM) in the oocytes and eggs of B. germanica. Whether oocytes accumulate CaM for immediate use or use at a later stage in their development is still unknown. We show that isolated follicle cells accumulate more CaM transcripts per unit RNA than any other control tissue. CaM transcript increases exponentially 4800-fold in follicles during the 96-hr vitellogenic period in the absence of cell division. This includes a 32-fold increase in total follicle RNA during the period and an 150-fold increase in relative titer of the CaM transcript. In comparison, levels of actin transcripts increase exponentially 1200-fold during the same developmental period. On the other hand fat body tissue shows little relative increase of CaM transcripts despite a 4-fold increase in total RNA over the 4-day developmental period. Both the CaM and actin transcripts are more highly concentrated in the Day 4 follicle cell layer, being found in 84- and 33-fold greater titer, respectively, than in fat body RNA. Deprivation of juvenile hormone (JH), by head ligation, not only causes atresia of the follicles, but also reduces accumulated CaM transcripts. Reestablishing JH titer by injection allows a selected population of follicles to develop to full size and also reinstates CaM transcript levels above that of unligated controls within 24 hr.

Comparison of defolliculated oocytes and intact follicles of the cockroach using the vibrating probe to record steady currents.

Follicle cells were removed by dissection from early vitellogenic oocytes of the cockroach Blattella germanica. The vibrating probe was used to record steady currents from 19 defolliculated oocytes and 19 intact follicles of the same developmental stage. Defolliculated oocytes generated currents that were stable and distinguishable (by intensity or selective direction) from background reference values. Distributions of the intensities of reference values and experimental values were, in general, similar in both intact and defolliculated preparations. The patterns of currents generated by preparations recorded in the mid-sagittal plane were analyzed for both defolliculated oocytes (n = 8) and intact follicles (n = 10). The larger, generally more mature preparations in both groups generated patterns of current similar to the pattern seen in mid-vitellogenic follicles (focused inward near the germinal vesicle (GV), the presumptive ventral side, and broadly outward on the apo-GV side, the presumptive dorsal side). Smaller sized preparations in both groups showed inward or outward current on the apo-GV aspect and, typically, inward current at the GV. Only two defolliculated oocytes, and no intact follicles, appeared to generate outward current at the GV, and we believe this observation resulted from recording slightly outside the mid-sagittal plane. We conclude that preparations during early-vitellogenesis initially generate currents without an asymmetric pattern and that the inward flux at the GV is the first step in developing patterns of currents. The results suggest that the oocyte (and not the follicle cell epithelium) is responsible for generating the various patterns of currents observed in early-vitellogenic stages. At the end of early-vitellogenesis, the follicle cell epithelium begins to adhere tightly to the oocyte. The possibility is considered that the follicle cells may influence the currents generated during mid-vitellogenesis.

Most egg calmodulin is a follicle cell contribution to the cytoplasm of the Blattella germanica oocyte.

A high concentration of calmodulin (CaM) appears in mid- to late vitellogenic cockroach follicles which composes 1.5% of total protein. CaM levels during oogenesis were estimated by densitometric analysis of immunoblots using anti-Blattella germanica egg CaM antibody as a probe. CaM accumulates in the follicle throughout the yolk deposition phase maintaining its highest level of accumulation during the later 6-fold increase in oocyte volume. Evidence suggests that this later accumulated CaM is synthesized by the follicle cells and deposited in the oocyte. In vitro experiments with [35S]-met showed that the highly abundant CaM accumulating in vitellogenic follicles may not all be synthesized by the oocyte. Isolated follicle cells incorporate 13-fold more [35S]-met into CaM than the oocytes themselves but do not accumulate the product. The follicle cells are capable of producing all the CaM observed in newly ovulated eggs. No CaM is detectable in transit in the hemolymph of the female. These facts argue that CaM produced by follicle cells is the most likely source of CaM in the vitellogenic oocyte. Indirect immunofluorescent staining with anti-egg CaM demonstrated that in early- and midvitellogenic follicles CaM is localized in the cytoplasm of follicle cells and the cytoplasmic compartment surrounding yolk granules of oocytes but is excluded from yolk granules. Immunofluorescence was most intense in the cortex of the oocyte and outside the membranes of yolk granules. Transport of CaM into the cytoplasmic compartment of the oocyte is possible without invoking traditional adsorptive endocytosis.

Models of pattern formation in insect oocytes.

Pattern formation in early insect development is dominated by coordination of the germ lines polarity with the polarity of the follicle cell layer. The production of an elaborate protective chorion, covering the ovulated oocyte, has made establishing parallel polarity of germ line and soma absolutely essential. Genetics and molecular biology, particularly on Drosophila melanogaster, have identified numerous signals passed from follicle cell to oocyte and vice versa. The physiological basis of this communication is beginning to be established with the identification of several membrane receptors and potential signal transduction steps. The contributions of three physiological models of pattern formation are discussed as they relate to the growing genetic model. Evidence for and against ionic currents as factors in polarity determinations is particularly emphasized.

The effect of ions, ion channel blockers, and ionophores on uptake of vitellogenin into cockroach follicles.

Since calcium plays an important role in vitellogenin binding and uptake in Nauphoeta cinerea and because calcium channels have been described in follicles of this species, we investigated the effect of various ions, ionophores, and ion channel blockers on vitellogenin uptake in vitro. Calcium significantly stimulated vitellogenin uptake; this effect could be substituted best by barium and less well by strontium and magnesium. The stimulatory effect of calcium, and to a certain extent also that of barium, was dependent on the vitellogenin concentration, whereas the effect of strontium and magnesium was not. In the presence of calcium, vitellogenin uptake was inhibited by barium, strontium, and magnesium as well as by the transition elements nickel, cobalt, and zinc, but not by manganese which had a stimulatory effect. Valinomycin, verapamil, tetraethylammonium, and atropine reduced vitellogenin uptake, while amiloride and ouabain were ineffective. Our results indicate that calcium inward (and possibly potassium outward) fluxes play an important role in vitellogenin uptake.

Patterns of ionic currents around the developing oocyte of the German cockroach, Blattella germanica.

The development of patterns of current around vitellogenic oocytes of the cockroach, Blattella germanica, was examined by means of a two-dimensional vibrating probe. Previtellogenic oocytes exhibited small unstable currents. Shortly after vitellogenic uptake began (oocytes 0.6-0.8 mm anterior to posterior) currents were either all inward or all outward at the plane of measurement. A dorsoventral pattern of currents was first observed around oocytes a little larger than 0.8 mm. Current exited dorsally (source) and entered ventrally (sink). In these oocytes source and sink were small, less than half the anterior-posterior length. As oocytes grew, relative sizes of source and sink increased until they extended across the major part of dorsal and ventral surfaces. Many late vitellogenic oocytes had a pattern of dorsal outward current with a bimodal distribution. At the onset of chorionation measured currents were again small, unstable, and exhibited no well-defined pattern. Current density was greatest during midvitellogenesis.

Cleaning insect oocytes by dissection and enzyme treatment.

Vitellogenic oocytes from the cockroach Blattella germanica were dissected free of the surrounding follicle cell layer. The continued presence of a resting potential indicated that the defolliculated oocytes remained viable. In other experiments, lysozyme was injected into last nymphal instars; scanning electron micrographs showed that the bacteroids normally found between the follicle cell epithelium and the oocyte membrane were removed by this treatment. Oocytes from injected animals were known to be viable because they developed into living progeny. To assess the mode of action of lysozyme, defolliculated oocytes were exposed in vitro to lysozyme and then a hypotonic rinse; scanning electron micrographs showed that these oocytes were largely free of bacteroids.

Modeling Currents About Vitellogenic Oocytes of the Cockroach, Blattella germanica.

An insect oocyte with a disc-shaped sink of current on its ventral surface during a vitellogenic phase of development is used to demonstrate modeling of expected currents arising from a mathematically defined source. A simple model with an analytical solution for the expected currents is compared to a more complex model in which expectations are computed by mathematical integration. The currents about two oocytes of different pattern type are interpreted in light of the two models.