Red Light-Dependent CO(2) Uptake and Oxygen Evolution in Guard Cell Protoplasts of Vicia faba L.: Evidence for Photosynthetic CO(2) Fixation

Suspensions of dark-adapted guard cell protoplasts of Vicia faba L. alkalinized their medium in response to irradiation with red light. The alkalinization peaked within about 50 minutes and reached steady state shortly thereafter. Simultaneous measurements of O₂ concentrations and medium pH showed that oxygen evolved in parallel with the red light-induced alkalinization. When the protoplasts were returned to darkness, they acidified their medium and consumed oxygen. Both oxygen evolution and medium alkalinization were inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). In photosynthetically competent preparations, light-dependent medium alkalinization is diagnostic for photosynthetic carbon fixation, indicating that guard cell chloroplasts have that capacity. The striking contrast between the responses of guard cell protoplasts to red light, which induces alkalinization, and that to blue light, which activates proton extrusion, suggests that proton pumping and photosynthesis in guard cells are regulated by light quality.     

Identification of early proteins induced by highly oncogenic human adenovirus 12 during lytic infection and in hamster tumors

“Early” proteins (EP) induced by infection of cultured human KB cells with human adenovirus 12 (Ad12) were resolved by electrophoresis on sodium dodecyl sulfate (SDS)-polyacrylamide slab gels. Ad12 infected cells were incubated either in the presence of cycloheximide (CH) (to enhance the synthesis of early mRNA relative to cell mRNA) or cytosine arabinoside (ara C) (to prevent the expression of late viral genes) for 8 hr and the proteins were labeled with [${}^{35}\text{S}$] methionine (met) in an isotonic (110 mM NaCl) or hypertonic medium (210 mM NaCl) in the presence of ara C. Seven Ad12 induced EPs with apparent molecular weights of 60,000 (60K), 16.5K, 15K, 13K, 12.5K, 11K, and 10K (EP1 to EP7, respectively) have been identified when infected cells were labeled in an isotonic medium with CH pretreatment or in hypertonic medium with or without CH pretreatment. Sera from hamsters bearing Ad12 induced tumors (Ad12 T antisera) immunoprecipitated at least four of these polypeptides.     

Hypertonicity-induced intracellular pH changes in rat mast cells

In a non-isotonic environment, cells can shrink or swell and return to their normal shape by activating ion transport pathways. Changes in intracellular pH (pHi) after osmotic stress have been identified in several cells. In order to study the mechanisms that regulate cytosolic pH of rat mast cells in a hypertonic medium, we used the pH sensitive dye, BCECF. Under these hypertonic conditions, pHi undergoes an alkalinization following an initial acidification. The alkalinization is mediated by a Na+/H+ exchanger, since it is inhibited by amiloride and lack of extracellular sodium. Under these conditions, the alkalinization is increased with the PKC activators, TPA and OAG, and partially blocked with trifluoperazine, an unspecific protein kinase C (PKC) and Ca2+ calmodulin-dependent protein kinases (Ca2+/CaM K) inhibitor. There is also an anion exchanger, blocked with DIDS but not activated by PKC, that participates in the observed alkalinization. However, Na+/H+ exchanger is the main mechanism involved in the alkalinization of pHi of mast cells in a hyperosmotic environment.     

Shrinkage-induced Activation of the Na+/H+ Exchanger in Ehrlich Ascites Tumor Cells: Mechanisms Involved in the Activation and a Role for the Exchanger in Cell Volume Regulation

Amiloride-sensitive, Na⁺-dependent, DIDS-insensitive cytoplasmic alkalinization is observed after hypertonic challenge in Ehrlich ascites tumor cells. This was assessed using the fluorescent pH-sensitive probe 2′,7′-bis-(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). A parallel increase in the amiloride-sensitive unidirectional Na⁺ influx is also observed. This indicates that hypertonic challenge activates a Na⁺/H⁺ exchanger. Activation occurs after several types of hypertonic challenge, is a graded function of the osmotic challenge, and is temperature-dependent. Observations on single cells reveal a considerable variation in the shrinkage-induced changes in cellular pH _i , but the overall picture confirms the results from cell suspensions. Shrinkage-induced alkalinization and recovery of cellular pH after an acid load, is strongly reduced in ATP-depleted cells. Furthermore, it is inhibited by chelerythrine and H-7, inhibitors of protein kinase C (PKC). In contrast, Calyculin A, an inhibitor of protein phosphatases PP1 and PP2A, stimulates shrinkage-induced alkalinization. Osmotic activation of the exchanger is unaffected by removal of calcium from the experimental medium, and by buffering of intracellular free calcium with BAPTA. At 25 mm HCO⁻ ₃, but not in nominally HCO⁻ ₃-free medium, Na⁺/H⁺ exchange contributes significantly to regulatory volume increase in Ehrlich cells. Under isotonic conditions, the Na⁺/H⁺ exchanger is activated by ionomycin, an effect which may be secondary to ionomycin-induced cell shrinkage. Received: 2 March 1995/Revised: 29 September 1995     

Hypertonicity induced apoptosis in HL-60 cells in the presence of intracellular potassium

Cell shrinkage is a hallmark of apoptosis. Potassium efflux, which is involved in cell shrinkage, has been previously described as an essential event of apoptosis. This study was designed to address the importance of potassium efflux in hypertonicity (450 mOsm and 600 mOsm) induced apoptosis. We initiated apoptosis in HL-60 cells in hypertonic medium consisting of either high concentrations of NaCl, mannitol or KCl. Apoptotic activity was evaluated based on the DNA content of the cells, annexin-V staining and calcium content. Apoptosis was initiated in hypertonic conditions consisting of high intracellular K⁺. We demonstrate that apoptosis can occur in the presence of high intracellular potassium contrary to previous predictions.     

The mechanism of intracellular acidification induced by glucose in Saccharomyces cerevisiae

Addition of glucose or fructose to cells of Saccharomyces cerevisiae adapted to grow in the absence of glucose induced an acidification of the intracellular medium. This acidification appeared to be due to the phosphorylation of the sugar since: (i) glucose analogues which are not efficiently phosphorylated did not induce internal acidification; (ii) glucose addition did not cause internal acidification in a mutant deficient in all the three sugar-phosphorylating enzymes; (iii) fructose did not affect the intracellular pH in a double mutant having only glucokinase activity; (iv) glucose was as effective as fructose in inducing the internal pH drop in a mutant deficient in phosphoglucose isomerase activity; and (v) in strains deficient in two of the three sugar-phosphorylating activities, there was a good correlation between the specific glucose- or fructose-phosphorylating activity of cell extracts and the sugar-induced internal acidification. In addition, in whole cells any of the three yeast sugar kinases were capable of mediating the internal acidification described. Glucose-induced internal acidification was observed even when yeast cells were suspended in growth medium and in cells suspended in buffer containing K+, which supports the possible signalling function of the glucose-induced internal acidification. Evaluation of internal pH by following fluorescence changes of fluorescein-loaded cells indicated that the change in intracellular pH occurred immediately after addition of sugar. The apparent Km for glucose in this process was 2 mM. Changes in both the internal and external pH were determined and it was found that the internal acidification induced by glucose was followed by a partial alkalinization coincident with the initiation of H+ efflux. This reversal of acidification could be due to the activity of the H+-ATPase, since it was inhibited by diethylstilboestrol. Coincidence between internal alkalinization and the H+ efflux was also observed after addition of ethanol.     

Anisotonic media and glutamate-induced ion transport and volume responses in primary astrocyte cultures

1. The responses of primary monolayer astrocyte cultures prepared from neonatal rat brains to hyper- and hypotonic media and to the addition of L-glutamic acid were examined as part of a systematic approach to use these cultures to obtain information on the mechanisms of the volume changes seen in astroglial cells in situ. 2. Addition of 200 mM mannitol to the medium to make it hypertonic caused cell shrinkage as measured with [14C]3-O-methyl-D-glucose, and also activated K+ and Cl- uptake measured with 86Rb+ and 36Cl- respectively. The increased ion uptake was completely inhibited by 0.1 mM bumetanide, showing that the Na+ + K+ + 2 Cl- co-transport system was being activated by cell shrinkage. 3. Studies of 86Rb+ uptake as a function of external K+ and hypertonic media showed a complex pattern. Increased bumetanide-sensitive, hypertonic-stimulated uptake of 86Rb+ was seen up to 20 mM K+0, with maximum stimulation being first reached at around 2 to 5 mM K+. At concentrations greater than 20 mM K+0 there was a further increase in bumetanide-sensitive 86Rb+ uptake, but there was no stimulation of this uptake by hypertonicity. There were also increases in bumetanide-insensitive 86Rb+ fluxes at [K+]0 higher than 20 mM that may have been due to opening of voltage-dependent K+ channels; this increased 86Rb+ flux was decreased in hypertonic medium. 4. When primary astrocyte cultures were swollen in hypotonic medium there was a rapid increase in volume as measured with [14C] 3-O-methyl-D-glucose, which then decreased in the continued presence of hypotonic medium. Thus, these cells exhibit volume regulatory decrease or RVD, as described for other cells. The possible ionic bases of this phenomenon have not yet been fully examined but the initial RVD did not appear to stimulate a furosemide-sensitive cotransport system. 5. Glutamate has been implicated as a possible endogenous effector of volume change in astrocytes. In the presence of ouabain, L-glutamate led to swelling of cultured astrocytes and increased uptake of 22Na+ and 36Cl-. It is suggested that this is due to uptake of L-glutamate with cotransport of Na+ and Cl-. Increased uptake was also seen for 86Rb+ in the absence of ouabain, and this was not seen in the absence of Na+.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of low electrolyte media on salt loss and hemolysis of mammalian red blood cells.

Cation loss and hemolysis of various mammalian red cells suspended in isotonic non-electrolyte media were investigated. Sucrose buffered with 10 mM Tris-Hepes, pH 7.4 was used as the non-permeable non-electrolyte. Mammals from which the red cells were derived include the human, guinea pig, rat, rabbit, newborn calf, newborn piglet and pig, all of which contain K as the predominant cation species (HK type) and the dog, cat, sheep and cow, all of which possess Na as the predominant cation species (LK type). Of HK cells, a rapid efflux of K takes place from humans, rats and guinea pigs. Of LK type cells, the dog and cat exhibit an augmented membrane permeability to Na. The governing factors which influence cation permeability are the change in pH, temperature, and ionic strength. In response to increase in pH, the red cells of humans, dogs and cats become more permeable to cations, whereas the red cells of rat and rabbit are unaffected. In response to increase in temperature, HK type cells exhibit augmented K efflux, while the Na loss from the dog and cat cells manifest a well-defined maximum at near 37 degrees C. In all cases, a small substitution of sucrose by an equal number of osmoles of salts results in a dramatic decrease in cation loss. By contrast, the red cells of the rabbit, newborn calf, adult cow, newborn piglet, adult pig and sheep display no discernible increase in ion-permeability under the conditions alluded to above. In some species including the newborn calf, dog, and cat, an extensive hemolysis occurs usually within an hour in isotonic buffered sucrose solution. The osmolarity of sucrose solution affects these cells differently in that as the osmolarity increases from 200--500 mM, hemolytic rates of the calf and dog reach a saturation near 300 mM sucrose, whereas the hemolytic rate of the cat decreases progressively. Common features pertaining to this hemolysis are (1) the intracellular alkalinization process; and (2) the diminution of the cell volume which take place prior to and onset of hemolysis. SITS, a potent anion transport inhibitor, completely protects the cells from hemolysis by inhibiting chloride flux and the concomitant rise in intracellular pH.     

The effect of osmotic pressure on the mobility and metabolism of human spermatozoa]

When human sperm was suspended in a hypertonic medium, their motility is damaged and their oxygen consumption is reduced. Hypotonic conditions are more disastrous because the cells become immotile. These results are discussed with reference to high osmolarity of seminal fluid.     

Solute-dependent activation of cell motility in strongly hypertonic solutions in <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis>, human melanoma HTB-140 cells and walker 256 carcinosarcoma cells

Published data concerning the effects of hypertonicity on cell motility have often been controversial. The interpretation of results often rests on the premise that cell responses result from cell dehydration, i.e. osmotic effects. The results of induced hypertonicity on cell movement of Dictyostelium discoideum amoebae and human melanoma HTB-140 cells reported here show that: i) hypertonic solutions of identical osmolarity will either inhibit or stimulate cell movement depending on specific solutes (Na⁺ or K⁺, sorbitol or saccharose); ii) inhibition of cell motility by hypertonic solutions containing Na⁺ ions or carbohydrates can be reversed by the addition of calcium ions; iii) various cell types react differently to the same solutions, and iv) cells can adapt to hypertonic solutions. Various hypertonic solutions are now broadly used in medicine and to study modulation of gene expression. The observations reported suggest the need to examine whether the other responses of cells to hypertonicity can also be based on the solute-dependent cell responses besides cell dehydration due to the osmotic effects.     

Role of Ionic Fluxes in the Apoptotic Cell Death of Cultured Cerebellar Granule Neurons

Cultured cerebellar granule neurons (CGC) increase survival in a medium containing 25 mM KCl (K25), and they die apoptotically when cultures are treated with staurosporine (St) or are transferred to a 5-mM KCl containing medium (K5). Apoptotic CGC show nuclear condensation and caspase-3 activation. Cell death induced by these conditions was partially prevented when cultures were maintained under alkaline conditions, which also induced a marked reduction of the caspase-3 activation. The acidification of the medium further increased cell death induced by both stimuli. Cultures transferred to K5 suffered an immediate intracellular alkalinization that remained constant during the time K5 was present. In contrast, St did not modify cytosolic pH at any of the evaluated times. On the other hand, DIDS, furosemide, and bumetanide prevented CGC death induced by K5 and St. Other drugs such as amiloride, EIPA, tamoxifen, NEM, or NPPB did not modify cell death induced by these conditions. Both DIDS and bumetanide markedly inhibited the processing and activation of caspase-3, and DIDS prevented the nuclear condensation induced by K5 and St. These findings suggest that pH is a condition that could contribute to the modulation of cell death induced by some stimuli and that other ions, such as potassium, could have a role in the initial phase of apoptotic death of CGC.     

Potassium extrusion by the moderately halophilic and alkaliphilic methanogen methanolobus taylorii GS-16 and homeostasis of cytosolic pH.

Methanolobus taylorii GS-16, a moderately halophilic and alkaliphilic methanogen, grows over a wide pH range, from 6.8 to 9.0. Cells suspended in medium with a pH above 8.2 reversed their transmembrane pH gradient (delta pH), making their cytosol more acidic than the medium. The decreased energy in the proton motive force due to the reversed delta pH was partly compensated by an increased electric membrane potential (delta psi). The cytosolic acidification by M. taylorii at alkaline pH values was accompanied by K+ extrusion. The cytosolic K+ concentration was 110 mM in cells suspended at pH 8.7, but it was 320 mM in cells suspended at neutral pH values. High external K+ concentrations (210 mM or higher) inhibited the growth of M. taylorii at alkaline pH values, perhaps by preventing K+ extrusion. Cells suspended at pH 8.5 and 300 mM external K+ failed to acidify their cytosol. The key observation indicative of the involvement of K+ transport in cytosolic acidification was that valinomycin (0.8 microM), a K+ uniporter, inhibited the growth of M. taylorii only at alkaline pH values. Experiments with resting cells indicated that at alkaline pH values valinomycin uncoupled catabolic reactions from ATP synthesis. Thus, K+/H+ antiport activity was proposed to account for the K+ extrusion and the uncoupling effect of valinomycin at alkaline pH values. Such antiport activity was demonstrated by the sharp drop in pH of the bulk medium of the cell suspension upon the addition of 0.1 M KCl. The antiporter appeared to be active only at alkaline pH values, which was in accordance with a possible role in pH homeostasis by M. taylorii growing at alkaline pH values.     

Hemolysis of human red blood cells by freezing and thawing in solutions containing polyvinylpyrrolidone: relationship with postthypertonic hemolysis and solute movements

An investigation was made into the effects of the presence of polyvinylpyrrolidone (PVP) on changes in human red blood cells suspended in hypertonic solutions, on posthypertonic hemolysis, and on freezing at temperatures down to ?12 °C.PVP is very effective at reducing hemolysis when the red blood cells are frozen at temperatures down to ?12 °C. However, the membranes of the cells recovered on thawing have become very permeable to sodium and potassium ions and there is a much increased hemolysis if the cells are resuspended in an isotonic solution of sodium chloride.The presence of PVP does not affect the dehydration of the cells or the development of a change in membrane permeability when the cells are shrunken in hypertonic solutions at 0 °C. Neither does its presence in the hypertonic solution reduce the extent of posthypertonic hemolysis at 4 °C (as measured by the hemolysis on resuspension in an isotonic solution of sodium chloride), but it is more effective than sucrose at reducing hemolysis when present in the resuspension solution. It is concluded that the PVP is able to prevent swelling and hemolysis of cells which are very permeable to cations by opposing the colloid osmotic pressure due to the hemoglobin. However, this does not explain how PVP is able to protect cells against freezing damage at high cooling rates, and a mechanism by which it might do this is discussed.     

Regulation by cell volume of Na+-K+-2Cl− cotransport in vascular endothelial cells: role of protein phosphorylation

Summary Na⁺-K⁺-2Cl^– cotransport in aortic endothelial cells is activated by cell shrinkage, inhibited by cell swelling, and is responsible for recovery of cell volume. The role of protein phosphorylation in the regulation of cotransport was examined with two inhibitors of protein phosphatases, okadaic acid and calyculin, and a protein kinase inhibitor, K252a. Both phosphatase inhibitors stimulated cotransport in isotonic medium, with calyculin, a more potent inhibitor of protein phosphatase I, being 50-fold more potent. Neither agent stimulated cotransport in hypertonic medium. Stimulation by calyculin was immediate and was complete by 5 min, with no change in cell Na + K content, indicating that the stimulation of cotransport was not secondary to cell shrinkage. The time required for calyculin to activate cotransport was longer in swollen cells than in normal cells, indicating that the phosphorylation step is affected by cell volume. Activation of cotransport when cells in isotonic medium were placed in hypertonic medium was more rapid than the inactivation of cotransport when cells in hypertonic medium were placed in isotonic medium, which is consistent with a shrinkage-activated kinase rather than a shrinkage-inhibited phosphatase. K252a, a nonspecific protein kinase inhibitor, reduced cotransport in both isotonic and hypertonic media. The rate of inactivation was the same in either medium, indicating that dephosphorylation is not regulated by cell volume. These results demonstrate that Na⁺-K⁺-2Cl^– cotransport is activated by protein phosphorylation and is inactivated by a Type I protein phosphatase. The regulation of cotransport by cell volume is due to changes in the rate of phosphorylation rather than dephosphorylation, suggesting the existence of a volume-sensitive protein kinase. Both the kinase and the phosphatase are constitutively active, perhaps to allow for rapid changes in cotransport activity.This work was supported by a Clinical Investigator Award DK01643 (to W.C.O) and a Grant-in-Aid from the American Heart Association of Georgia.     

Evidence for differences among the sectors of potentially lethal damage expressed by hypertonic treatment in plateau-phase V79 cells after exposure to neutrons or gamma rays: the importance of distinction between alpha and beta-PLD forms

Expotentially growing and plateau-phase V79 cells were exposed to various doses of neutrons and plated either immediately or after treatment in hypertonic medium (250-500 mM NaCl) to express radiation-induced potentially lethal damage (PLD). Postirradiation treatment of exponentially growing cells in hypertonic medium (500 mM) resulted in a decrease in both Dq and D0, whereas postirradiation treatment of plateau-phase cells in hypertonic medium (in the range between 200 to 1,500 mM) resulted mainly in a reduction of Dq. This difference in response between exponentially growing and plateau-phase cells may reflect differences in the chromatin structure in cells at various stages of the cell cycle, affecting fixation of radiation-induced damage. Exposure of plateau-phase cells to gamma rays, on the other hand, resulted in a treatment time and salt concentration-dependent decrease in Dq along with a decrease in D0. Repair of neutron-induced, hypertonic treatment-sensitive PLD, measured by delaying treatment for various periods after irradiation, was found to proceed with a t1/2 of about 1 h. This is similar to the repair kinetics obtained by delaying treatment of plateau-phase cells with 150 microM beta-D-arabinofuranosyladenine (araA) after exposure to gamma rays or neutrons and contrasts the repair kinetics observed after exposure of cells to gamma rays. In this case, hypertonic treatment was found to affect a form of PLD repaired with a t1/2 of 10-15 min (beta-PLD) and araA, a different form of PLD, repaired with a t1/2 of about 1 h (alpha-PLD). Based on these results it is hypothesized that the sector of lesions affected by hypertonic treatment and araA coincides after exposure to neutrons (effect on alpha-PLD) but only partly overlaps after exposure to gamma rays (due to the effect on beta-PLD of hypertonic treatment). The results presented, together with previously published observations, suggest a differential induction and/or fixation by hypertonic medium of the alpha- and beta-PLD forms as the LET of the radiation increases. Furthermore, they indicate that direct comparison of the effects of a postirradiation treatment, as well as of the repair kinetics obtained by its delayed application after exposure to radiations of various LET, should be made with caution.     

Creatine and creatinine transport in old and young human red blood cells

The time course of creatine influx or efflux as measured in populations of red cells or red cell ghosts with normal age distribution does not follow simple two-compartment kinetics. This suggests that the contributions of individual cells to transport as measured in the populations as a whole are not uniform. In agreement with this inference, fractionation of red cell populations with respect to cell age shows that transport in young cells is considerably faster than in old cells.The dependence of creatine transport on creatine concentration in the medium follows an equation that can be interpreted to represent a super-imposition of a saturable component (apparent K_m = 0.02 mM) and another component that cannot be saturated up to a creatine concentration of 5.0 mM. In contrast to the non-saturable component, the saturable component depends on the energy metabolism of the cell and can be inhibited by β-guanidinopropionic acid and the proteolytic enzyme pronase. This latter finding suggests that the saturable component represents active transport that is mediated by a transport protein. The non-saturable component is little, if at all, dependent on cell age while the saturable component is higher in young cells than in old cells. Phloretin inhibits both components of creatine flux, but the maximal inhibition that can be achieved at high concentration is only 70–80%.Under the experimental conditions used for the study of creatine transport, creatinine equilibration between cells and medium follows the kinetics expected for a steady-state two-compartment system. Creatinine flux is proportional to creatine concentration over the concentration range studied (up to 5 mM). It cannot be inhibited by β-guanidinopropionic acid or pronase.     

Different patterns of cell volume regulation in hyposmotic media between attached and suspended HeLa cells.

Both attached and suspended HeLa cells swelled in a medium of a hypotonic osmolality of 235 mosmol/kg H2O. When the osmolality was further decreased to 166 mosmol/kg H2O, attached cells instantly swelled and then rapidly lost water and K+, followed by slow gains of them. Suspended cells instantly swelled and then K+ loss and regulatory volume decrease (RVD) occurred. Neither 0.1 mM ouabain nor 10 mM TEA changed the water loss of attached cells, whereas ouabain inhibited RVD of suspended cells. Quinine (1 mM) inhibited water losses from both cells and comparison of the losses implies stronger activation of K+ channel in attached cells than in suspended cells. Omission of medium Ca2+ or addition of 10 mM BaCl2 inhibited RVD in part. These results suggest that hyposmotic stress induces net water loss from attached cells, associated with K+ release through the Ca(2+)-dependent K+ channel. Suspended cells osmotically swell, followed by RVD with K+ and Na+ releases through the K+ channel and Na(+)-pump, respectively. The different patterns of volume changes may relate to the difference of activity or time of activation of the K+ channel between both cells.     

Glycolysis of red cells suspended in solutions of impermeable solutes. Intracellular pH and glycolysis.

The glycolytic rate human red cells suspended in a sucrose medium of low or physiological pH was higher than that of the cells suspended in Ringer's medium of the same. pH. The medium pHP-glycolytic rate curve of red cells suspended in soucrose media shifted to the acidic side by about one unit compared with that of cells suspended in Ringer's medium. Similarly, the pattern of glycolytic intermediates in red cells suspended in a sucrose medium resembled that in cells suspended in Ringer's solution of about one unit higher pH. These phenomena could be ascribed to the change of intracellular pH, which was measured by the 5,5'-dimethyl-oxazolidine-2,4-dione method. A similar stimulation of glycolysis was observed when sodium citrate was added to red cells suspended in Ringer's solution at constant pH. These observations indicate that membrane-impermeable non-electrolytes or anions stimulate glycolysis of red cells by elevation ofthe intracellular pH. Red cell glycolysis is influenced mainly by the intracellular pH rather than by the pH of the suspending medium.     

Nitrate uptake and extracellular alkalinization by the green alga Hydrodictyon reticulatum in blue and red light

Nitrate uptake and the medium alkalinization related to it were studied with nets of the coenocytic, giant cell, green alga Hydrodictyon reticulatum. A comparison of red, blue and white light irradiation showed no special control of nitrate uptake and of the corresponding alkalinization of the external medium by light quality, but rather a response as expected for the photosynthetic apparatus. In the dark, nitrate uptake rates amounted to one-fifth of those in saturating white light. This is in contrast to the chlorococcal microalga Monoraphidium braunii, where blue light specifically switched on nitrate uptake-dependent alkalinization and where uptake and reduction of nitrate strongly depended on blue light; the rates in pure red light and in the dark being very low. The stoichiometric ratio between nitrate taken up and extracellular alkalinization was close to 1 (0.86) in air with CO2 but close to 2 (1.84) in N2 for nitrate pre-loaded cells. In the absence of any carbon source, a high proportion of the absorbed and reduced nitrogen is released, most of it as ammonium which causes the excess alkalinization and some as nitrite, which lowers the ratio. Nitrite and ammonium release rates under anaerobic, CO2-free conditions were also independent of red or blue light and continued for several hours when the medium was buffered at pH 6. The data indicate that nitrate uptake, but less its reduction, is regulated differently in vacuolate, coenocytic algae from microalgae. In Hydrodictyon, nitrate uptake and reduction seems to be controlled by energy supply; in various microalgae, in addition, it is controlled specifically by blue light.     

Induction of a Ca(2+)-activated K+ channel in human erythrocytes by mechanical stress.

Mechanical deformation of normal ATP-replete human erythrocytes increased their permeability to Ca2+ sufficiently to turn on the Ca(2+)-activated K+ channel (the Gardos channel). When Ca2+ is absent, mechanical deformation of normal erythrocytes induces an equivalent increase the permeability of both Na+ and K+, In the presence of 0.1 to 1 mM Ca2+, a further increase in the K+ efflux rate was seen. There was no increase in Na+ flux above that induced by deformation itself. The involvement of the Ca(2+)-activated H channel was verified by showing the specific inhibitors of the channel, quinine and charybdotoxin, prevent the Ca(2+)-induced increase in K+ efflux. These results are consistent with a model of sickle cell dehydration proposed by Bookchin et al. ((1987) Prog. Clin. Biol. Res. 240, 193-200). The estimated rate of Ca2+ entry under these conditions (37 degrees C, 1000 dyne/cm2, and laminar shear) was about 1 mmol/loc per h.