Turgor-dependent unloading of photosynthates from coats of developing seed of Phaseolus vulgaris and Vicia faba. Turgor homeostasis and set points

Key physiological characteristics of turgor-dependent efflux of photosynthates were examined using excised coats and cotyledons of developing Phaseolus vulgaris (cv. Redland Poineer) and Vicia faba (cv. Coles Prolific) seed during the linear phase of seed fill. Exposure to solutions of high osmotic potential inhibited net uptake of [¹⁴C]sucrose by cotyledons at developmental stages less than 60% of their final dry weight. The effect could not be fully reversed by transferring cotyledons to solutions set at lower osmotic potentials. The inhibition became apparent at osmotic potentials that were higher than those that caused stimulation of efflux from seed coats. Net [¹⁴C]sucrose uptake by cotyledons at more advanced stages of development was unaffected by external osmotic potential. Specified tissue layers were removed from seed coats by pretreatment with pectinase. Efflux studies with the pectinase-modified coats of Phaseolus and Vicia seed demonstrated that the cellular site of turgordependent efflux was the ground parenchyma and thin-wall parenchyma transfer cells, respectively. Coats subjected to long-term (hours) incubations, under hypo-osmotic conditions, exhibited the capacity for turgor regulation. This was mediated by turgor-dependent efflux of solutes. The solutes exchanged were of nutritional significance to the developing embryo. The relationship between efflux and coat turgor was characterised by a turgor-independent phase at low turgors. Once turgor exceeded a minimal value (set point), efflux increased in proportion to the magnitude of the turgor deviation (error signal) from the set point. For coats of sink-limited seed of Vicia and Phaseolus, efflux exhibited apparent saturation at turgors above 0.25 and 0.5 MPa respectively. The putative turgor set point and slope of the turgor-dependent component of efflux varied with seed development, the prevailing source/sink ratio and genetic differences in seed growth rate. The nature of these kinetic variations was compatible with the competitive ability of the seed. A turgor homeostat model is proposed that incorporates the observed functional attributes of turgor-dependent efflux. Operationally, the model provides a mechanistic basis for the integration of assimilate demand by the cotyledons with assimilate import into and unloading from the seed coat.     

Osmotic regulation of assimilate unloading from seed coats of Vicia faba. Role of turgor and identification of turgor-dependent fluxes

Osmotic regulation of assimilate efflux from excised coats of developing Vicia faba (cv. Coles Prolific) seed was examined by exposing these to bathing solutions (adjusted to –0. 02 to –0. 75 MPa with sorbitol) introduced into the cavity vacated by the embryo. ¹⁴C photosynthate efflux was found to be independent of solution osmotic potentials below –0. 63 MPa. At higher osmotic potentials, efflux was stimulated and exhibited a biphasic response to osmotic potential with apparent saturation being reached at –0. 37 MPa. Efflux could be repeatedly stimulated and slowed by exposing seed coats to solutions of high and low osmotic potentials, respectively. Manipulation of components of tissue water potential, with slowly- and rapidly-permeating osmotica, demonstrated that turgor functioned as the signal regulating ¹⁴C photosynthate efflux. Com-partmental analysis of ¹⁴C photosynthate preloaded seed coats was consistent with exchange from 4 kinetically-distinct compartments. The kinetics of turgor-dependent efflux exhibited characteristics consistent with the transport mechanism residing in the plasma membranes of the unloading cells. These characteristics included the rapidity (<2 min) of the efflux response to turgor increases, similar rate constants for efflux from the putative turgor-sensitive and cytoplasmic compartments and the apparent small pool size from which turgor-dependent efflux could repeatedly occur. In contrast, influx of [¹⁴C] sucrose across the plasma and tonoplast membranes was found to be insensitive to turgor. The plasma membrane [¹⁴C] sucrose influx was unaffected by p-chloromercuribenzenesulfonic acid and erythrosin B and exhibited a linear dependence on the external sucrose concentration. This behaviour suggested that influx across the plasma membrane occurs by passive diffusion. Preloading excised seed coats with a range of solutes demonstrated that turgor-dependent efflux exhibited partial solute selectivity. Based on these findings, it is proposed that turgor controls assimilate exchange from the seed coat by regulating an efflux mechanism located in the plasma membranes of the unloading cells.     

Cellular pathway of photosynthate transport in coats of developing seed of Vicia faba L. and Phaseolus vulgaris L. II. Principal cellular site(s) of efflux

The cells responsible for the photosynthate efflux from coatsof developing seed of Vicia faba L. and Phaseolus vulgaris L.were elucidated using known properties of the efflux mechanism.Sensitivity of sucrose efflux to NEM and high potassium concentrationswas retained by seed-coat halves of Phaseolus following pectinaseremoval of the branch parenchyma cell layer. In contrast, removalof the thin-walled parenchyma transfer cell layer from Viciaseed-coat halves abolished this sensitivity. The membrane-impermeantthiol-binding fluorochrome, qBBr, selectively stained the surfaceof the thin-walled parenchyma transfer cells. This phenomenonwas inhibited by the slowly permeable sul-phydryl agent, PCMBS,indicating that the plasma membranes of these cells are enrichedin sulphydryl groups characteristic of membrance porter proteins.On the basis that carrier-mediated sucrose efflux from seedcoats appears to be proton coupled, the putative plasma membraneH+-ATPase was used as a marker for the cells responsible forcarrier-mediated photosynthate efflux. When seed-coat halveswere exposed briefly at pH 8.5 to the weak acid fluorochrome,SRG, the ground parenchyma and thin-walled parenchyma transfercell layers selectively accumulated the dye. The apparent lowpH environment in the walls of these cells that renders SRGmembrane permeant appeared to be maintained by a VAN-sensitiveproton pump. The findings with SRG were corroborated by thecyto-chemical localization of plasma membrane ATPase activityto the ground parenchyma and thin-walled parenchyma transfercells using precipitation of cerium phosphate. Together, ourobservations provide qualified support for the conclusion thatcarrier-mediated photosynthate efflux from coats of Phaseolusand Vicia seed is primarily restricted to the ground parenchymaand thin-walled parenchyma transfer cell layers, respectively. Key words: Ground parenchyma, Phaseolus vulgaris L., photosynthate efflux, seed coat, transfer cell, Vicia faba L.     

Proton extrusion in seed coats of Phaseolus vulgaris L.

Abstract. The present investigations were designed to identify proton pumps in seed coats of Phaseolus vulgaris L. Vacated seed-coat halves were exposed to bathing solutions with indicators for proton pump action and the pH changes in the media were measured. Fusicoccin increased the rate of proton extrusion from the seed coats. Orthovanadate and abscisic acid retarded the proton extrusion evoked by fusicoccin. Abolition of the proton extrusion by parachloromercuriphenylsulphonic acid was partially reversed by diethioerythritol. The extrusion was stimulated by high osmolarities (100 mol m⁻³ sorbitol), potassium ions (100 mol m⁻³ KCI) and light. Old seed coats reacted more rapidly to fusicoccin treatments than young ones. Proton pumping in seed coats and cotyledons showed differential responses to fusicoccin, K⁺ and sucrose. In contrast to seed coats, medium acidification by cotyledons was prohibited by addition of sucrose. The significance of proton pumps for photosynthate transfer in vivo is discussed.     

Quantitative Analysis of Photosynthate Unloading in Developing Seeds of Phaseolus vulgaris L. : II. Pathway and Turgor Sensitivity

Phloem import and unloading in perfused bean (Phaseolus vulgaris L.) seed coats were investigated using steady-state labeling. Though photosynthate import and unloading were significantly reduced by perfusion, measurements of photosynthate fluxes in perfused seed coats proved useful for the study of unloading mechanisms in vivo. Phloem import was stimulated by lowered seed coat cell turgor, as demonstrated by an increase in tracer and sucrose import to seed coats perfused with high concentrations of an osmoticum. The partitioning of photosynthates between retention in the seed coat and release to the perfusion solution also was turgor sensitive; increases in seed coat cell turgor stimulated photosynthate release to the apoplast at the expense of photosynthate retention within the seed coat. There was no evidence of a turgor-sensitive sucrose uptake mechanism in perfused seed coats. Thus, the turgor sensitivity of photosynthate partitioning within perfused seed coats was consistent with a turgor-sensitive efflux control mechanism. Measurements of tracer equilibration and sugar partitioning in perfused seed coats provided strong evidence for symplastic phloem unloading in seed coats.     

Efflux of photosynthate and acid from developing seed coats of Phaseolus vulgaris L.: a chemiosmotic analysis of pump-driven efflux

Seed coats of Phaseolus vulgaris L. unload photosynthetic products,mineral ions and acid into the apoplastic space surroundingthe embryo. We report measurements, on detached seed coats,of the rates of unloading of photosynthates, ions and acid atdifferent external pH and in the presence of treatments intendedto alter the rate of proton pumping. We also report measurementsof membrane potential difference (PD) and of cytoplasmic pHunder the same conditions, measurements which have allowed usto validate the treatments we used and to investigate functionalrelationships between membrane processes. A chemiosmotic model of the seed-coat cell membrane is proposed,in which sucrose efflux and acid efflux are both driven by theproton pump. Sucrose efflux is proposed to occur by sucrose/protonantiport driven by the proton-motive force (PMF), and acid effluxto occur by pumped protons accompanied by a passive efflux ofanions. We use our measurements to estimate the net efflux ofsucrose on the antiporter and the total efflux of protons onthe pump. We have tested the model by using experimental treatments designedto manipulate the pump rate as the independent variable. Underthese conditions, and assuming the model is correct, the pumprate determines the cytoplasmic pH. Over the range covered byour experiments the net sucrose efflux is dependent on externaland cytoplasmic pH, the latter having the major role. The effluxof acid, under the same treatments, depends primarily on theproton pump rate, and was found to be well fitted by a quadraticfunction of pump rate. This means that, as pump rate increases,an increasing proportion of the pump output is used by acidefflux and a decreasing proportion by sucrose antiport. The membrane PD, although an important component of the PMF,does not appear to function in rate control of net sucrose orof acid efflux, since neither efflux is correlated with membranePD under our treatments which vary the pump rate. The PD correlateswell with external potassium concentration, and seems largelydetermined by the diffusion of potassium ions and anions. Key words: Phaseolus vulgaris L, photosynthate efflux, proton pump, sucrose/proton antiport, seed coat, membrane transport model     

Osmotic regulation of assimilate unloading from seed coats of Vicia faba. Assimilate partitioning to and within attached seed coats.

The significance of the osmotic potential of the seed apoplast sap as a regulator of assimilate transfer to and within coats of developing seed of Vicia faba (cv. Coles Prolific) was assessed using attached empty seed coats and intact developing seed. Following surgical removal of the embryos, through windows cut in the pod walls and underlying seed coats, the resulting attached “empty” seed coats were filled with solutions of known osmotic potentials (–0. 02 versus –0. 75 MPa). Sucrose efflux from the coats was elevated at the higher osmotic potential (high osmotic concentration) for the first 190 min of exchange. Thereafter, this efflux was depressed relative to efflux from coats exposed to the low osmotic potential (high osmotic concentration) solution. This subsequent reversal in efflux was attributable to an enhanced diminution of the coat sucrose pools at the high external osmotic potential. Indeed, when expressed as a proportion of the current sucrose pool size, relative efflux remained elevated for coats exposed to the high osmotic potential solution. Measurement of potassium and sucrose fluxes to and from their respective pools in the coat tissues demonstrated that the principal, fluxes, sensitive to variative in the external osmotic potential, were phloem import into and efflux from the “empty” coats. Phloem import, consistent with a pressure-driven phloem transport mechanism, responded inversely with changes in the external osmotic potential. In contrast, sucrose and potassium efflux from the coats exhibited a positive dependence on the osmotic potential. Growth rates of whole seed were approximately doubled by enclosing selected pods in water jackets held at temperatures of 25°C. compared to 15°C. The osmotic potential of sap collected from the seed apoplast remained constant and independent of the temperature-induced changes in seed growth rates and hence phloem import. Based on these findings, it is proposed that control of phloem import by changes in the external osmotic potential observed with “empty” seed coats has no significance as a regulator of assimilate import by intact seed. Rather, maintenance of the seed apoplast osmotic potential, independent of seed growth rate, suggests that the observed osmotic regulation of efflux from the coats may play a key role in integrating assimilate demand by the embryo with phloem import.     

Changes in Photosynthate Unloading from Perfused Seed Coats of Phaseolus vulgaris L. Induced by Osmoticum and Ethylenediaminetetraacetate (EDTA)

Photosynthate unloading in Phaseolus vulgaris L. seed coatswas studied by treating perfused seed coats with differing concentrationsof an osmoticum and ethylenediaminetetraacetate (EDTA). Largechanges in osmoticum concentration typically produced rapidchanges in efflux of unlabelled sugar and steady-state-labelled¹⁴C-photosynthate. Osmoticum-induced changes in photosynthateefflux were caused by phloem import stimulation at low cellturgor and net efflux stimulation by high cell turgor. Eventhough rapid changes in sugar and tracer efflux were often inducedby osmoticum treatments, the specific activity of sugar releasedfrom seed coats was not greatly affected by these treatmentsand was similar to the specific activity of sugar remainingin the seed coat after perfusion. Thus, tracer was transportedfrom the phloem throughout the seed coat sugar pool before itwas released to the apoplast. This result is most consistentwith symplastic phloem unloading throughout perfused seed coats,because apoplastic transport between cells within the seed coatwas blocked by perfusion. Photosynthate efflux was stimulatedby simultaneous treatment of seed coats with EDTA and differentconcentrations of an osmoticum; loss of photosynthate from seedcoats did not appear to be tissue-specific. Key words: Phaseolus vulgaris, seed coat, photosynthate unloading, turgor, EDTA     

Turgor-dependent unloading of assimilates from coats of developing legume seed. Assessment of the significance of the phenomenon in the whole plant

The in vivo significance of turgor-dependent unloading was evaluated by examining assimilate transport to and within intact developing seeds of Phaseolus vulgaris (cv. Redland Pioneer) and Vicia faba (cv. Coles Prolific). The osmotic potentials of the seed apoplast were low. As a result, the osmotic gradients to the seed coat symplast were relatively small (i.e. 0.1 to 0.3 MPa). Sap concentrations of sucrose and potassium in the seed apoplast and coat symplast accounted for some 45 to 60% of the osmotic potentials of these compartments. Estimated turnover times of potassium and sucrose in the seed apoplast of < 1 h were some 5 to 13 times faster than the respective turnover times in the coat symplast pools. The small osmotic gradient between the seed apoplast and coat symplast combined with the relatively rapid turnover of solutes in the apoplast pool, confers the potential for a small change in assimilate uptake by the cotyledons to be rapidly translated into an amplified shift in the cell turgor of the seed coat. Observed adjustments in the osmotic potentials of solutions infused between the coat and cotyledons of intact seed were consistent with the in vivo operation of turgor-dependent unloading of solutes from the coat. Homeostatic regulation of turgor-dependent unloading was indicated by the maintenance of apoplast osmotic potentials of intact seeds when assimilate balance was manipulated by partial defoliation or elevating pod temperature. In contrast, osmotic potentials of the coat symplast adjusted upward to new steady values over a 2 to 4 h period. The resultant downward shift in coat cell turgor could serve to integrate phloem import into the seed coat with the new rates of efflux to the seed apoplast. Circumstantial evidence for this linkage was suggested by the approximate coincidence of the turgor changes with those in stem levels of ³²P used to monitor phloem transport. The results obtained provide qualified support for the in vivo operation of a turgor homeostat mechanism. It is proposed that the homeostat functions to integrate assimilate demand by the cotyledons with efflux from and phloem import into the coats of developing legume seed.     

Differential effects of turgor on sucrose and potassium transport at the tonoplast and plasma membrane of sugar beet storage root tissue

When turgor was increased, by decreasing the concentration of mannitol bathing discs of sugar beet storage root tissue, the rates of sucrose and potassium uptake into the vacuole were decreased. At all external mannitol concentrations the rate of sucrose and potassium uptake across the plasma membrane was an order of magnitude greater than the rate of quasi-steady uptake into the vacuole, implying a very large efflux. Efflux of both sucrose and potassium was increased at high turgor. However, while increasing turgor decreased the rate of K⁺ uptake, the rate of sucrose uptake at the plasma membrane increased with time. Compartmental analysis of tracer exchange kinetics was used to determine unidirectional K⁺ fluxes. From these results, it was estimated that the increase in K⁺ efflux accompanying a 1.5 MPa increase in turgor could lead to a net increase of 140mol^?3h^?1 in the external potassium concentration. It is suggested that the turgor-imposed increase in solute efflux is a means of regulating intracellular osmotic pressure and/or turgor in sugar beet storage roots, but that sucrose is preferentially retrieved from the apoplast, even under conditions of excessively high turgor. However, much of this sucrose is probably lost from the cell, implying a ‘futile’ sucrose transport cycle at the plasma membrane. The turgor-stimulated leak of potassium could play a major role in the regulation of turgor pressure in sugar beet storage root tissue.     

Sucrose Release into the Endosperm Cavity of Wheat Grains Apparently Occurs by Facilitated Diffusion across the Nucellar Cell Membranes

Nutrients required for the growth of the embryo and endosperm of developing wheat (Triticum aestivum L.) grains are released into the endosperm cavity from the maternal tissues across the nucellar cell plasma membranes. We followed the uptake and efflux of sugars into and out of the nucellus by slicing grains longitudinally through the endosperm cavity to expose the nucellar surface to experimental solutions. Sucrose uptake and efflux are passive processes. Neither was sensitive to metabolic inhibitors, pH, or potassium concentration. p-Chloromercuribenzene sulfonate, however, strongly inhibited both uptake and efflux, although not equally. Except for p-chloromercuribenzene sensitivity, these characteristics of efflux and the insensitivity of Suc movement to turgor pressure are similar to those of sucrose release from maize pedicels, but they contrast with legume seed coats. Although the evidence is incomplete, movement appears to be carrier mediated rather than channel mediated. In vitro rates of sucrose efflux were similar to or somewhat less than in vivo rates, suggesting that transport across the nucellar cell membranes could be a factor in the control of assimilate import into the grain.     

Sugar retrieval by coats of developing seeds of Phaseolus vulgaris L. and Vicia faba L

Influxes of glucose, fructose and sucrose were characterised for coat cells of developing seeds of Phaseolus vulgaris L. and Vicia faba L. by monitoring uptake of [(14)C]sugars into excised seed-coat halves and two different protoplast populations derived from seed coats. Sugar influxes by the two populations of protoplasts were similar for each sugar species [sucrose > (fructose approximately glucose)] and hexoses competed with sucrose. Concentration-dependent influxes of all three sugars by excised seed coats could be described by a simple directly proportional relationship between concentration ([S]) and uptake rate (v) in the physiological range of sugar concentrations (v approximately A.[S]). Alternatively, with the exception of fructose influx by Vicia, all could be fitted to a Michaelis-Menten relationship, as could sucrose uptake by Vicia protoplasts. Apparent K(m) values were high ( approximately 100-500 mM) compared with those reported for other systems. Sucrose transport was distinct from glucose and fructose transport in both species. Sugar influx was decreased by p-chloromercuribenzenesulfonic acid, carbonylcyanide m-chlorophenylhydrazone and erythrosin B. These responses are consistent with sugar/H(+) symport acting to retrieve photoassimilates leaked to the apoplasm during post-sieve element transport within seed coats.     

Pulsing Cl- channels in coat cells of developing bean seeds linked to hypo-osmotic turgor regulation

Seed coat cells in the developing seeds of grain legumes release nutrients to the developing embryo. This occurs into an apoplastic space that separates the maternal (seed coat) and filial (embryo) generations. Protoplasts of seed coat cells from coats of Phaseolus vulgaris L. seeds were isolated and whole-cell current across their plasma membranes was characterized using the patch-clamp technique. A pulsing inward current that displayed a spontaneous activation and voltage-dependent inactivation was observed. The frequency and magnitude of the current pulses were positively dependent on cytoplasmic Cl(-) concentrations and independent of external cations. The pulse current was inhibited by DIDS and La(3+), but not by Gd(3+). Single channel events (conductance=18 pS) could be identified with the inactivating phase of the pulses. Together, these findings are consistent with the current being carried by a burst of Cl(-) efflux through Cl(-)-permeable channels that activate almost simultaneously. Neomycin caused a reversible inhibition of the pulsed current, suggesting that its activation is likely to be modulated by an IP(3)-dependent intracellular Ca(2+) release. The pharmacological profiles of Cl(-) efflux from excised seed coats were comparable with those of the Cl(-) channels in the whole cell configuration, suggesting that the Cl(-) channels may underpin Cl(-) efflux from the seed coats. Efflux of Cl(-) from the seed coats was also stimulated by hypo-osmotic treatment as was the frequency and magnitude of Cl(-) channel in whole-cell patch clamp experiments. This implies that the Cl(-) channels responsible for the pulsed Cl(-) currents are likely to be a component of the turgor-regulatory mechanism in developing bean seeds.     

A suite of sucrose transporters expressed in coats of developing legume seeds includes novel pH-independent facilitators

A suite of newly discovered sucrose transporter genes, PsSUF1, PsSUF4, PvSUT1 and PvSUF1, were isolated from the coats of developing pea (Pisum sativum L.) and bean (Phaseolus vulgaris L.) seeds. Sequence analysis indicated that deduced proteins encoded by PsSUF1, PvSUT1 and PvSUF1 clustered in a separate sub-group under sucrose transporter Clade I, whereas the deduced protein encoded by PsSUF4 clustered in Clade II. When expressed in yeast, these genes were shown to encode sucrose transporters with apparent Michaelis Menten constant (Km) values ranging from 8.9 to 99.8 mm. PvSUT1 exhibited functional characteristics of a sucrose/H+ symporter. In contrast, PsSUF1, PvSUF1 and PsSUF4 supported the pH- and energy independent transport of sucrose that was shown to be bi-directional. These transport properties, together with that of counter transport, indicated that PsSUF1, PvSUF1 and PsSUF4 function as carriers that support the facilitated diffusion of sucrose. Carrier function was unaffected by diethylpyrocarbonate and by maltose competition, suggesting that the sucrose binding sites of these transporters differed from those of known sucrose/H+ symporters. All sucrose transporters were expressed throughout the plant and, of greatest interest, were co-expressed in cells considered responsible for sucrose efflux from seed coats. The possible roles played by the novel facilitators in sucrose efflux from seed coats are discussed.     

Cellular pathway of photosynthate transport in coats of developing seed of Vicia faba L. and Phaseolus vulgaris L. I. Extent of transport through the coat symplast

The extent of post-phloem solute transport through the coatsymplasts of developing seeds of Vicia faba L. and Phaseolusvulgaris L. was evaluated. For Vicia seed coats, the membrane-impermeantfluorochrome, CF, moved radially from the chalazal vein to reachthe chlorenchyma and thin-walled parenchyma transfer cell layers.Thereafter, the fluorochrome moved laterally in these two celllayers around the entire circumference of the seed coat. Transferof CF from the chalazal vein was inhibited by plasmolysis ofattached ‘empty’ seed coats. In contrast, the spreadof phloem imported CF was restricted to the ground parenchymaof Phaseolus seed coats. Fluorochrome loaded into the outermostground parenchyma cell layer was rendered immobile followingplasmolysis of excised seed-coat halves. Phloem-imported [¹⁴C]sucroseand the slowly membrane permeable sugar, L-[¹⁴C]glucose, werepartitioned identically between the vascular and non-vascularregions of intact Vicia seed coats. For ¹⁴C-photosynthates,these partitioning patterns in attached ‘empty’Vicia seed coats were unaffected by PCMBS, but inhibited byplasmolysis. Tissue autoradiographs of intact Phaseolus seedcoats demonstrated that a pulse of ¹⁴C-photosynthate moved fromthe veins to the grounds tissues. In excised Vicia seed coats,preloaded with ¹⁴C-photosynthates, the cellular distributionof residual ¹⁴C-label was unaffected by PCMBS. In contrast,PCMBS caused the ¹⁴C-photosynthate levels to be elevated inthe veins and ground parenchyma relative to the branch parenchymaof Phaseolusseed coat halves. Based on the above findings, itis concluded that the phloem of Vicia seed coats is interconnectedto two major symplastic domains; one comprises the chlorenchyma,the other the thin-walled parenchyma plus thin-walled parenchymatransfer cells. For Phaseolusseed coats, the phloem forms amajor symplastic domain with the ground parenchyma. Key words: Phaseolus vulgaris L, phloem unloading, photosynthate transport, seed coat, symplast, Vicia faba L     

Swelling-activated Taurine and Creatine Effluxes from Rat Cortical Astrocytes are Pharmacologically Distinct

Primary cultures of rat cortical astrocytes undergo a swelling-activated loss of taurine and creatine. In this study, the pharmacological characteristics of the taurine and creatine efflux pathways were compared, and significant differences were shown to exist between the two. Both taurine and creatine effluxes were rapidly activated upon exposure of astrocytes to hypo-osmotic media, and rapidly inactivated upon their return to iso-osmotic media. The relative rates of taurine and creatine efflux depended upon the magnitude of the hypo-osmotic shock. Anion-transport inhibitors strongly inhibited taurine efflux, with the order of potency being NPPB > DIDS > niflumic acid. DIDS and NPPB had less of an inhibitory effect on creatine efflux, whereas tamoxifen and niflumic acid actually stimulated creatine efflux. These data are consistent with separate pathways for taurine and creatine loss during astrocyte swelling.     

Transport Systems of Ventricaria ventricosa: Asymmetry of the Hyper- and Hypotonic Regulation Mechanisms

Hyper- and hypotonic stresses elicit apparently symmetrical responses in the alga Ventricaria. With hypertonic stress, membrane potential difference (PD) between the vacuole and the external medium becomes more positive, conductance at positive PDs (Gmpos) increases and KCl is actively taken up to increase turgor. With hypotonic stress, the membrane PD becomes more negative, conductance at negative PDs (Gmneg) increases and KCl is lost to decrease turgor. We used inhibitors that affect active transport to determine whether agents that inhibit the K(+) pump and hypertonic regulation also inhibit hypotonic regulatory responses. Cells whose turgor pressure was held low by the pressure probe (turgor-clamped) exhibited the same response as cells challenged by hyperosmotic medium, although the response was maintained longer than in osmotically challenged cells, which regulate turgor. The role of active K(+) transport was confirmed by the effects of decreased light, dichlorophenyldimethyl urea and diethylstilbestrol, which induced a uniformly low conductance (quiet state). Cells clamped to high turgor exhibited the same response as cells challenged by hypo-osmotic medium, but the response was similarly transient, making effects of inhibitors hard to determine. Unlike clamped cells, cells challenged by hypo-osmotic medium responded to inhibitors with rapid, transient, negative-going PDs, with decreased Gmneg and increased Gmpos (linearized I-V), achieving the quiet state as PD recovered. These changes are different from those exerted on the pump state, indicating that different transport systems are responsible for turgor regulation in the two cases.     

Potassium channels in primary cultures of seawater fish gill cells. II. Channel activation by hypotonic shock

Previous studies performed on apical membranes of seawater fish gills in primary culture have demonstrated the existence of stretch-activated K(+) channels with a conductance of 122 pS. The present report examines the involvement of K(+) channels in ion transport mechanisms and cell swelling. In the whole cell patch-clamp configuration, K(+) currents were produced by exposing cells to a hypotonic solution or to 1 microM ionomycin. These K(+) currents were inhibited by the addition of quinidine and charybdotoxin to the bath solution. Isotopic efflux measurements were performed on cells grown on permeable supports using (86)Rb(+) as a tracer to indicate potassium movements. Apical and basolateral membrane (86)Rb effluxes were stimulated by the exposure of cells to a hypotonic medium. During the hypotonic shock, the stimulation of (86)Rb efflux on the apical side of the monolayer was inhibited by 500 microM quinidine or 100 microM gadolinium but was insensitive to scorpion venom [Leirus quinquestriatus hebraeus (LQH)]. An increased (86)Rb efflux across the basolateral membrane was also reduced by the addition of quinidine and LQH venom but was not modified by gadolinium. Moreover, basolateral and apical membrane (86)Rb effluxes were not modified by bumetanide or thapsigargin. There is convincing evidence for two different populations of K(+) channels activated by hypotonic shock. These populations can be separated according to their cellular localization (apical or basolateral membrane) and as a function of their kinetic behavior and pharmacology.     

Lamprothamnium, a Euryhaline Charophyte: IV. MEMBRANE POTENTIAL, IONIC FLUXES AND METABOLIC ACTIVITY DURING TURGOR ADJUSTMENT

The early time-course of turgor adjustment following a hyper-or hypo-osomotic shock was examined in the brackish-water charophyteLamprothamnium papulosum. The response to a reduction in turgorwas a five to seven-fold stimulation of the influxes of Cl^–,K⁺ and Na⁺. The distribution of radioactive tracers in the cellsuggested that the ionic composition of the cytoplasm was strictlycontrolled during turgor adjustment. Metabolic activity wasrelatively unaffected by the loss of turgor. high fluxes throughthe cytoplasm, and a cytoplasmic K^– concentration possiblyas high as 280 mol m^–3. Osmotic adjustment to a lower salinity was achieved by largeincreases in the passive effluxes of K⁺ and Cl^– ratherthan by decreases in their influxes. The membrane remained hyperpolarized during hyperosmotic adjustmentbut depolarized after a hypo-osmotic change. This result isdiscussed in relation to changes in the driving forces for ionmovements during osmotic transitions. Key words: Lamprothamnium, Turgor, Osmotic stress