Fluxes of 86Rb+ in “isolated” guard cells of Vicia faba L.

Steady-state ⁸⁶Rb⁺ fluxes and contents were measured in stomatal guard cells of Vicia faba L., using isolated epidermal strips in which all cells other than the guard cells had been killed by a brief ultrasound treatment. Flux experiments were carried out for a range of stomatal apertures, achieved by incubation in light or dark, or on solutions containing different concentrations of RbCl (1–30 mM). At pH 5.5 the efflux curve of ⁸⁶Rb⁺ could be fitted to the sum of two exponential terms and it was possible to calculate compartmental contents and fluxes. The data indicate that steady-state stomatal opening is achieved by regulation of both influx and efflux. Salt accumulation was not always adequate to contribute the observed osmotic requirement for opening, and this may indicate that some other solute accounts for the discrepancy.Abbreviations and Symbols A amplitude - k rate constant - Mes 2-(N-morpholino)-ethanesulphonic acid - Q_T, Q_C, Q_V total, cytoplasmic, vacuolar chemical content - Q^* tracer content - Ø_P, Ø_l plasmalemma, tonoplast fluxes This work was supported by a Research Studentship from the Science and Engineering Research Council. I thank Professor E.A.C. MacRobbie for much helpful discussion and advice.     

Volume-induced increase of K+ and Cl− permeabilities in Ehrlich ascites tumor cells. Role of internal Ca2+

Summary Ehrlich ascites tumor cells resuspended in hypotonic medium initially swell as nearly perfect osmometers, but subsequently recover their volume within 5 to 10 min with an associated KCl loss. 1. The regulatory volume decrease was unaffected when nitrate was substituted for Cl^–, and was insensitive to bumetanide and DIDS. 2. Quinine, an inhibitor of the Ca²⁺-activated K⁺ pathway, blocked the volume recovery. 3. The hypotonic response was augmented by addition of the Ca²⁺ ionophore A23187 in the presence of external Ca²⁺, and also by a sudden increase in external Ca²⁺. The volume response was accelerated at alkaline pH. 4. The anti-calmodulin drugs trifluoperazine, pimozide, flupentixol, and chlorpromazine blocked the volume response. 5. Depletion of intracellular Ca²⁺ stores inhibited the regulatory volume decrease. 6. Consistent with the low conductive Cl^– permeability of the cell membrane there was no change in cell volume or Cl^– content when the K⁺ permeability was increased with valinomycin in isotonic medium. In contrast, addition of the Ca²⁺ ionophore A23187 in isotonic medium promoted Cl^– loss and cell shrinkage. During regulatory volume decrease valinomycin accelerated the net loss of KCl, indicating that the conductive Cl^– permeability was increased in parallel with and even more than the K⁺ permeability. It is proposed that separate conductive K⁺ and Cl^– channels are activated during regulatory volume decrease by release of Ca²⁺ from internal stores, and that the effect is mediated by calmodulin.     

Genetics of esterases in Drosophila. V. Characteristics of the “pupal” esterase in D. virilis

From analysis of the properties of the pupal esterase (p-esterase) in Drosophila virilis, it is concluded that it is heat stable, its electrophoretic detection depends on culture density, its expression is stage specific, and it is not a variant of esterase 2. It was also demonstrated that p-esterase, like esterase 6, is activated by injections of the juvenile hormone into larvae. Heat treatment of heat-resistant D. virilis stocks led to decreased activities of the juvenile hormone dependent esterases but did not affect those of the heat-sensitive stocks. It is suggested that heat resistance in D. virilis is related to some functional features of the system of modifier genes controlling the phenotypic expression of esterases.     

The sided action of Na+ and of K+ on reconstituted shark (Na+ + K+)-ATPase engaged in Na+−Na+ exchange accompanied by ATP hydrolysis. I. The ATP activation curve

The ATP hydrolysis dependent Na⁺-Na⁺ exchange of reconstituted shark (Na⁺ + K⁺)-ATPase is electrogenic with a transport stoichiometry as for the Na⁺-K⁺ exchange, suggesting that translocation of extracellular Na⁺ is taking place via the same route as extracellular K⁺. The preparation thus offers an opportunity to compare the sided action of Na⁺ and of K⁺ on the affinity for ATP in a reaction in which the intermediary steps in the overall reaction seems to be the same without and with K⁺. With Na⁺ but no K⁺ on the two sides of the enzyme, the ATP-activation curve is hyperbolic and the affinity for ATP is high. Extracellular K⁺ in concentrations of 50 μM (the lowest tested) and up gives biphasic ATP activation curves, with both a high- and a low-affinity component for ATP. Cytoplasmic K⁺ also gives biphasic ATP-activation curves, however, only when the K⁺ concentration is 50 mM or higher (Na⁺ + K⁺ = 130 mM). The different ATP-activation curves are explained from the Albers-Post scheme, in which there is an ATP-dependent and an ATP-independent deocclusion of E₂(Na₂⁺) and E₂(K₂⁺), respectively, and in which the dephosphorylation of E₂-P is rate limiting in the presence of Na⁺ (but no K⁺) extracellular, whereas in the presence of extracellular K⁺ it is the deocclusion of E₂(K₂⁺) which is rate limiting.     

Stimulation of H+transport activity of vacuolar H+ATPase by activation of H+PPase in Kalanchoë blossfeldiana

In Kalanchoë blossfeldiana cv. Tom Thumb the initial rate of ATP-dependent H+-transport into tonoplast vesicles was stimulated up to three times if the H+-ATPase (EC 3.6.1.3) was energized a few minutes after pre-energization of the H+-PPase (EC 3.6.1.1). H+-PPase-activated ATP-dependent H+-transport was observed in plants of K. blossfeldiana cultivated in short day (SD) or long day (LD) conditions expressing different degrees of crassulacean acid metabolism (CAM). However, based on the higher activity and protein amount of H+-PPase and H+-ATPase present in the vacuolar membrane of SD plants the maximum H+-transport activity in the stimulated mode of the H+-ATPase was significantly higher in tonoplast vesicles of SD plants than of LD plants. Hence, a co-ordinated action of the H+-PPase and H+-ATPase at the tonoplast of Kalanchoë could allow a higher transport capacity at the vacuolar membrane when plants perform high CAM. Immunoprecipitation experiments with an antiserum raised against the A-subunit of the vacuolar H+-ATPase of Mesembryanthemum crystallinum L. showed that in SD and LD plants of K. blossfeldiana the H+-PPase was co-precipitated with the vacuolar H+-ATPase holoenzyme. The co-percipitation of the two transport proteins indicates a close structural localization of the H+-PPase and the A-subunit of the vacuolar H+-ATPase.     

Localization of Ca2+-binding proteins of rat cortex on two-dimensional gels—II. analysis of Ca2+ binding proteins in ammonium sulfate fractions of rat brain

A total of seven high-affinity calcium-binding proteins have been detected in rat brain. This was accomplished using a combination of ammonium sulfate fractionation, two-dimensional gel electrophoresis, western blotting and ⁴⁵Ca²⁺-autoradiography. Of these seven proteins, three are detectable in a crude tissue punch of rat cortex while four are seen only after protein enrichment with ammonium sulfate. Four of the seven proteins detected in this study have been identified: calmodulin, the B subunit of calcineurin, the intestinal vitamin D-dependent calcium-binding protein and parvalbumin. The identities of the other three proteins visualized by ⁴⁵Ca²⁺-autoradiography in this study are unknown.     

Discrimination of Na+-independent transport systems L, T, and asc in erythrocytes. Na+ independence of the latter a consequence of cell maturation?

On the basis of inhibition analysis two bicyclic amino acid analogs appear to enter human red blood cells by much the same Na+-independent mediation, whereas striking differences are apparent in the routes for tryptophan and leucine, confirming a role for System T, but also suggesting the participation of a third system of low affinity somewhat selective for weakly basic amino acids. System T of the human cell is specifically inhibited by 4-azidophenylalanine, and is highly sensitive, relative to System L, to N-ethylmaleimide inhibition. Uptake by System T approaches its steady state much more slowly than does System L, and its participation in trans-stimulation is questionable, whereas that of System L is as usual strong. A different added transport system became apparent in the slow approach of the Na+-independent mediation of uptake of 3- and 4-carbon dipolar amino acids by the nucleated pigeon red cell to its steady state. In that cell System T makes at most a minor contribution. The patterns of trans-stimulation of fluxes among selected pairs of amino acids in the pigeon cell correspond to a usual participation in transmembrane exchange by System L, and also by the new transport system. An important but not the sole source of the heterogeneity in the pigeon cell is the participation of the system conspicuously involved in the transport of alanine, serine, and threonine, among other amino acids. This route of transport of these amino acids is made conspicuous by their small transport by other Na+-independent agencies, notably System L. Reactivity with this system is enhanced by a side change hydroxyl or sulfhydryl group. Uptake by this route as tested by threonine showed little inhibition by cysteinesulfinate under conditions inhibitory to System asc; also a sensitivity to lowering of pH unlike that seen with System asc. The new Na+-dependent transport system appears to be a species variant of quite similar Na+-independent systems discovered by Young et al. (Young, J. D., Ellory, J. C., and Tucker, E. M. (1975) Nature (Lond.) 254, 156-157; Fincham, D. A., Mason, D. K., and Young, J. D. (1982) Biochem. Soc. Trans. 11, 776-777) in sheep and horse erythrocytes on the basis of their absence in phenotypes. These authors have emphasized several similarities in these two cases to Na+-dependent System asc, and they propose that Na+ dependence has specifically been lost on maturation of the red cells without major changes in amino acid selectivity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Role of Na+–H+ exchange in the modulation of L-type Ca2+ current during fluid pressure in rat ventricular myocytes

Application of fluid pressure (FP) using pressurized fluid flow suppresses the L-type Ca²⁺ current through both enhancement of Ca²⁺ release and intracellular acidosis in ventricular myocytes. As FP-induced intracellular acidosis is more severe during the inhibition of Na⁺–H⁺ exchange (NHE), we examined the possible role of NHE in the regulation of I_Ca during FP exposure using HOE642 (cariporide), a specific NHE inhibitor. A flow of pressurized (∼16 dyn/cm²) fluid was applied onto single rat ventricular myocytes, and the I_Ca was monitored using a whole-cell patch-clamp under HEPES-buffered conditions. In cells pre-exposed to FP, additional treatment with HOE642 dose-dependently suppressed the I_Ca (IC₅₀ = 0.97 ± 0.12 μM) without altering current–voltage relationships and inactivation time constants. In contrast, the I_Ca in control cells was not altered by HOE642. The HOE642 induced a left shift in the steady-state inactivation curve. The suppressive effect of HOE642 on the I_Ca under FP was not altered by intracellular high Ca²⁺ buffering. Replacement of external Cl⁻ with aspartate to inhibit the Cl⁻-dependent acid loader eliminated the inhibitory effect of HOE642 on I_Ca. These results suggest that NHE may attenuate FP-induced I_Ca suppression by preventing intracellular H⁺ accumulation in rat ventricular myocytes and that NHE activity may not be involved in the Ca²⁺-dependent inhibition of the I_Ca during FP exposure.     

Regulation of Ca2+ Sparks by Ca2+ and Mg2+ in Mammalian and Amphibian Muscle. An RyR Isoform-specific Role in Excitation–Contraction Coupling?

Ca2+ and Mg2+ are important mediators and regulators of intracellular Ca2+ signaling in muscle. The effects of changes of cytosolic [Ca2+] or [Mg2+] on elementary Ca2+ release events were determined, as functions of concentration and time, in single fast-twitch permeabilized fibers of rat and frog. Ca2+ sparks were identified and their parameters measured in confocal images of fluo-4 fluorescence. Solutions with different [Ca2+] or [Mg2+] were rapidly exchanged while imaging. Faster and spatially homogeneous changes of [Ca2+] (reaching peaks >100 microM) were achieved by photolysing Ca NP-EGTA with laser flashes. In both species, incrementing cytosolic [Ca2+] caused a steady, nearly proportional increase in spark frequency, reversible upon [Ca2+] reduction. A greater change in spark frequency, usually transient, followed sudden increases in [Ca2+] after a lag of 100 ms or more. The nonlinearity, lag, and other features of this delayed effect suggest that it requires increase of [Ca2+] inside the SR. In the frog only, increases in cytosolic [Ca2+] often resulted, after a lag, in sparks that propagated transversally. An increase in [Mg2+] caused a fall of spark frequency, but with striking species differences. In the rat, but not the frog, sparks were observed at 4-40 mM [Mg2+]. Reducing [Mg2+] below 2 mM, which should enable the RyR channel's activation (CICR) site to bind Ca2+, caused progressive increase in spark frequency in the frog, but had no effect in the rat. Spark propagation and enhancement by sub-mM Mg2+ are hallmarks of CICR. Their absence in the rat suggests that CICR requires RyR3 para-junctional clusters, present only in the frog. The observed frequency of sparks corresponds to a channel open probability of 10(-7) in the frog or 10(-8) in the rat. Together with the failure of photorelease to induce activation directly, this indicates a basal inhibition of channels in situ. It is proposed that relief of this inhibition could be the mechanism by which increased SR load increases spark frequency.     

Importance of L3T4+ and Lyt-2+ cells in the immunologic control of infection with Mycobacterium bovis strain bacillus Calmette-Guérin in mice. Assessment by elimination of T cell subsets in vivo

The course of infection after injection of small doses of bacillus Calmette-Guérin (BCG) was studied in mice which were depleted in vivo of T cell subsets by administration of either anti-L3T4 or anti-Lyt-2 mAb. The results presented herein strongly suggest that the L3T4+ subpopulation play a pivotal role in the immunologic control of BCG infection because the depletion of L3T4+ cells led to a dramatic increase in the number of viable bacteria. Depletion of Lyt-2+ cells had no significant effect on the course of infection. These results were confirmed by using adoptive transfer experiments which showed that protective immunity was mediated by L3T4+ cells generated in the spleen as a result of infection. Moreover, T cells capable of controlling the recurrence of BCG multiplication from residual bacteria remaining in organs after the recovery from infection were shown to belong to the L3T4+ subpopulation.     

II. Selective accumulation of MPP+ in the substantia nigra: A key to neurotoxicity?

The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is rapidly metabolized to a 1-methyl-4-phenylpyridinium species (MPP+) in the squirrel monkey. After administration of toxic doses of MPTP, the concentration of MPP+ in the substantia nigra appears to increase during the first 72 hours, reaching the highest concentration of any central nervous system (CNS) tissue studied. In contrast, the concentration of this compound in other brain areas suggested time dependent elimination during the same period. Pretreatment of animals with the monoamine oxidase (MAO) inhibitor pargyline blocks both the neurotoxic action and the biotransformation of MPTP. In animals given pargyline and MPTP, initial MPTP levels are much higher in all brain regions than in those not receiving pargyline, but by 12 hours, MPTP levels had fallen rapidly in all regions except the substantia nigra and the eye. It may be that the selective toxicity of MPTP is related in some way to the accumulation of its oxidized metabolite in the substantia nigra.     

Cytoplasmic free calcium in Riccia fluitans L. and Zea mays L.: Interaction of Ca2+ and pH?

In cells of Zea mays (root hairs, coleoptiles) and Riccia fluitans (rhizoids, thalli) intracellular Ca²⁺ and pH have been measured with double-barrelled microelectrodes. Free Ca²⁺ activities of 109–187 nM (Riccia rhizoids), 94–160 nM (Riccia thalli), 145–231 nM (Zea root hairs), 84–143 nM (Zea coleoptiles) were found, and therefore identified as cytoplasmic. In a few cases (Riccia rhizoids), free Ca²⁺ was in the lower millimolar range (2.3±0.8 mM). A change in external Ca²⁺ from 0.1 to 10 mM caused an initial and short transient increase in cytoplasmic free Ca²⁺ which finally levelled off at about 0.2 pCa unit below the control, whereas in the presence of cyanide the Ca²⁺ activity returned to the control level. It is suggested that this behaviour is indicative of active cellular Ca²⁺ regulation, and since it is energy-dependent, may involve a Ca²⁺-ATPase. Acidification of the cytoplasmic pH and alkalinization of the vacuolar pH lead to a simultaneous increase in cytoplasmic free Ca²⁺, while alkalinization of pH_c decreased the Ca²⁺ activity. Since this is true for such remote organisms as Riccia and Zea, it may be concluded that regulation of cytoplasmic pH and free Ca²⁺ are interrelated. It is further concluded that double-barrelled microelectrodes are useful tools for investigations of intracellular ion activities in plant cells.Symbols and abbreviations _m, _m membrane potential difference, changes thereof - PVC polyvinylchloride     

Use of protoplasts in the improvement of filamentous microorganisms. I. Action of N-methyl-N′-nitro-N-nitrosoguanidine on protoplasts of Streptomycetes

Summary The effects of N-methyl-N–nitro-N-nitroso-guanidine (NTG) on protoplasts of Streptomycetes are markedly different from its action on spores, showing high mutagenic activity even at concentrations having no marked effect on protoplast survival. Strain improvement, eg in chlorotetracycline-producing strains of S. aureofaciens, was most effective when protoplasts were subjected to prolonged treatment (2 h) with low concentrations of NTG (50 /ug/ml).     

Diurnal rhythm of hypophyseal cells in mice. IV. Pars Distalis∗

Abstract

Using various staining techniques and methods, the following cells were differentiated from pars distalis of the pituitary gland in the mouse: corticotropic, somatotropic, lactotropic, thyrotropic and gonadotropic cells.

Diurnal activity of the differentiated cells in pars distalis of the pituitary gland was determined by the method of karyometry.

The karyometric analysis showed the existence of a distinct diurnal rhythm in nuclear volume in all five types of the differentiated cells in both sexes.