Kinetin-induced stimulation of electrogenic pumping in soybean suspension cultures is unrelated to signal transduction

Primary modes of action of cytokinins have been thought to involve stimulation of the electrogenic H⁺ pump and-or opening of plasmamembrane Ca²⁺ channels. In order to test these hypotheses, rapid changes in membrane transport in response to cytokinin application were studied in heterotrophic suspension-cultured callus of soybean (Glycine max (L.) Merr.) using electrophysiological techniques. Kinetin (N⁶-furfurylaminopurine; 2 M) elicited membrane hyperpolarization of 13±1 mV. This effect occurred even at membrane poteintials more negative than the most negative ionic equilibrium potential, and therefore might have resulted either from stimulation of the electrogenic pump, or from closure of ionic channels. The former mechanism of action appears most likely because (i) kinetin-induced membrane hyperpolarization is not accompanied by a significant change in plasma-membrane resistivity and (ii) hyperpolarization is abolished by cyanide, which inhibits electrogenic pump activity by depletion of cellular ATP.Electrogenic pumping is also activated by two other cytokinins: N⁶-(benzyl)adenine and trans-zeatin. However, it is unlikely that the hormonal effect on electrogenesis is directly related to transduction of the cytokinin signal, for the following reasons: (i) hormonally inactive, but chemically related compounds (cis-zeatin, adenine) also elicited membrane hyperpolarization; (ii) hormonally active, N⁹-substituted cytokinins failed to stimulate electrogenesis; (iii) the chemically unrelated cytokinin N,N-diphenylurea also failed to stimulate electrogenesis.The results imply that the kinetin effect on electrogenic pumping is related to adenine, or its metabolism, and not hormonal action. Adenine was absorbed by soybean cells, but not in sufficient quantities to have a significant effect on adeninenucleotide pools. It appears likely that the control of electrogenesis requires either the presence of a purine free base (i.e. no substituents at the N⁹ position) or phosphoribosylation of the free base. No evidence was found for cytokinin-induced Ca²⁺-channel opening, though it is argued that such an event might be physiologically relevant, yet undetectable with the methods employed. It is essential that future studies on cytokinin signal transduction — especially as they relate to membrane transport — take into account the possibility that metabolic effects unrelated to hormone action are dominant.Abbreviations and symbols bzl⁶Ade N⁶ (benzyl)adenine - SRB Soybean Recording Buffer - V_m membrane potential     

Sodium-transport NADH-quinone reductase of a marineVibrio alginolyticus

The respiratory chain of a marine bacterium,Vibrio alginolyticus, required Na⁺ for maximum activity, and the site of Na⁺-dependent activation was localized on the NADH-quinone reductase segment. The Na⁺-dependent NADH-quinone reductase extruded Na⁺ as a direct result of redox reaction. It was composed of three subunits, , , and , with apparentMr of 52, 46, and 32 KDa, respectively. The reduction of ubiquinone-1 to ubiquinol proceeded via ubisemiquinone radicals. The former reaction was catalyzed by the FAD-containing subunit. This reaction showed no specific requirement for Na⁺. For the formation of ubiquinol, the presence of the subunit and the FMN-containing subunit was essential. The latter reaction specifically required Na⁺ for activity and was strongly inhibited by 2-n-heptyl-4-hydroxyquinolineN-oxide. It was assigned to the coupling site for Na⁺ transport. The mode of energy coupling of redox-driven Na⁺ pump was compared with those of decarboxylase- and ATP-driven Na⁺ pumps found in other bacteria.     

Efficiency of proton extrusion by chemically modified mitochondria

Bovine heart mitochondria were treated with limited amounts of iodoacetamide, 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, phenylglyoxal, tetranitromethane and 1-fluoro-2,4-dinitrobenzene, respectively. Examination of the respiration and proton extrusion characteristics of the chemically modified mitochondria suggests that sulfhydryl and imidazole groups are not directly involved in proton pumping, but that some of the labeled carboxyl, amino, guanidinium and phenolic groups may participate in an indirect proton-extrusion process. Cross-linking mitochondria with glutaraldehyde drastically decreases the efficiency of proton extrusion, whereas treatment of mitochondria with valeraldehyde under similar conditions did not affect the proton-pumping efficiency significantly. The latter observations show that conformational change in the inner mitochondrial membrane may play a crucial role in the active translocation of protons coupled to electron transport. Comparison of the reactivities of the essential amino and carboxyl groups in mitochondria in different oxidation states suggests that these two types of essential functional groups are more exposed to water in the oxidized state. An indirect mechanism for proton pumping based on protein conformational change driven by electron transport based on the results of the present chemical modification studies is suggested.     

Nutrition ofPinus caribaea in its native savanna habitat

Summary Fertility levels in soils beneathPinus caribaea trees were examined in the Mountain Pine Ridge savannas, Belize, where fire control has precipitated the development of pine woodland. Slight surface soil enrichment was recorded beneath pine canopies, but to levels well below those found beneath associated hardwoods. Estimates of total nutrient pools beneath trees showed modest cation accumulation beneath a 73 year old tree but some defecits in Ca and Mg beneath a 24 year old tree. A tap root cutting experiment on trees of the same species revealed no significant declines in foliar nutrient levels after 19 months. It is concluded that no pronounced long-term deterioration in soil fertility levels is developing beneath stands ofP. caribaea in the savanna, although some temporary nutrient declines may exist beneath young pine stands. Atmospheric inputs are the most likely source of nutrient accretion and it is suggested that the establishment of hardwood associates with pine may enhance the rates of nutrient capture from this source.     

Bacteriorhodopsin-mediated photoelectric responses in lipid/water systems

Summary Bacteriorhodopsin-mediated photopotential generation has been studied in two kinds of lipid/water systems: (1) decane solution of asolectin was used as the lipid phase; (2) a mixture of bacteriorhodopsin sheets and hexane solution of phosphatidyl choline was applied onto a water surface to form a monolayer, and then the monolayer was covered with a 0.3-mm decane layer. In both cases, illumination was found to induce formation of an electric potential difference, with the bulk water phase being found negative when measured with a vibrating electrode. In the latter, but not in the former, system small amounts of a protonophorous uncoupler were found to stimulate the photoresponse. Large amounts of the uncoupler proved depressing in both systems. Phenyldicarbaundecaborane anion (PCB^–) was shown to substitute for the uncoupler, being much more potent both as an activator and as an inhibitor of the photoresponse. In both studied systems, gramicidin A inhibits the photoresponse, the effect being greatly potentiated by K⁺, Na⁺ or H⁺ ions.In the system decane solution of asolectin/water, an Ag/AgCl electrode immersed into the lipid phase can be used instead of a vibrating electrode. All the measured features of the photoelectric responses observed with any of these electrodes were found to be quite similar to those inherent in a phospholipid-impregnated collodion film adsorbing bacteriorhodopsin sheets on one of its surfaces.A scheme is discussed built on the assumption that photopotentials in all the studied systems are due to an uphill light-dependent transport of H⁺ ions from the bulk water phase to a water cavity localized between a bacteriorhodopsin sheet and the surface of the bulk lipid phase. Thus, the above lipid/water systems containing bacteriorhodopsin are composed of four, rather than two, phases, as was supposed previously.Bacteriorhodopsin-mediated photopotential generation has been studied also in the decane/water system without phospholipids. This system with bacteriorhodopsin sheets added to the water phase demonstrates a light-dependent photoelectric response reaching 1.5 V, which can be measured only by a vibrating electrode. The photoresponse starts after a lag period of several seconds. Switching off the light results in the reversal of the light-induced electric potential change. The off-effect also has a lag period. The action spectrum of the photoresponse shows at least two maxima: a smaller at 560 nm and a larger at <420 nm. Free retinal can substitute for bacteriorhodopsin in the studied system. All the above effects disappear if, instead of air, argon is used. In the system decane solution of asolectin/water, a slow photoelectric response of this type can be demonstrated at neutral pH in the presence of gramicidin and at pH 4 without gramicidin. A suggestion is put forward that the slow photoelectric response is due to an interface Volta-potential change induced by a product of photooxidation of bacteriorhodopsin and/or free retinal released from bacteriorhodopsin.     

Electrical properties of the plasma membrane of microplasmodia ofPhysarum polycephalum

Summary Microplasmodia ofPhysarum polycephalum have been investigated by conventional electrophysiological techniques. In standard medium (30mm K⁺, 4mm Ca⁺⁺, 3mm Mg⁺⁺, 18mm citrate buffer, pH 4.7, 22°C), the transmembrane potential differenceV _m is around –100 mV and the membrane resistance about 0.25 m².V _m is insensitive to light and changes of the Na⁺/K⁺ ratio in the medium. Without bivalent cations in the medium and/or in presence of metabolic inhibitors (CCCP, CN^–, N ₃ ^– ),V _m drops to about 0 mV. Under normal conditions,V _m is very sensitive to external pH (pH _o ), displaying an almost Nernstian slope at pH _o =3. However, when measured during metabolic inhibition,V _m shows no sensitivity to pH _o over the range 3 to 6, only rising (about 50 mV/pH) at pH _o =6. Addition of glucose or sucrose (but not mannitol or sorbitol) causes rapid depolarization, which partially recovers over the next few minutes. Half-maximal peak depolarization (25 mV with glucose) was achieved with 1mm of the sugar. Sugar-induced depolarization was insensitive to pH _o . The results are discussed on the basis of Class-I models of charge transport across biomembranes (Hansen, Gradmann, Sanders and Slayman, 1981,J. Membrane Biol. 63:165–190). Three transport systems are characterized: 1) An electrogenic H⁺ extrusion pump with a stoichiometry of 2 H⁺ per metabolic energy equivalent. The deprotonated form of the pump seems to be negatively charged. 2) In addition to the passive K⁺ pathways, there is a passive H⁺ transport system; here the protonated form seems to be positively charged. 3) A tentative H⁺-sugar cotransport system operates far from thermodynamic equilibrium, carrying negative charge in its deprotonated states.     

Inhibition of N-ethylmaleimide of the MgATP-driven proton pump of the chromaffin granules

The thiol reagent N-ethylmaleimide (NEM) completely inhibits the proton pump activity of the H+-ATPase in chromaffin granule 'ghosts' at concentrations which only partly (approximately 20%) inhibit the Mg2+-dependent ATP hydrolysis. Half-maximal inhibition was obtained at approximately 13 microM NEM as compared to 18 microM for the classical proton channel inhibitor N,N'-dicyclohexylcarbodiimide (DCCD), and the apparent stoichiometry of the inhibitors at complete inhibition was NEM : DCCD congruent to 1 : 2. HIgh concentrations of NEM (greater than 100 microM) induce a dissipation of the transmembrane potential generated by MgATP. These findings establish NEM as a valuable proton channel inhibitor in chromaffin granules and explain the rather complex effect of NEM previously reported for catecholamine accumulation in this organelle.     

Changes in Regional Brain Levels of Amino Acid Putative Neurotransmitters After Prolonged Treatment with t he Anticonvulsant Drugs Diphenylhydantoin, Phenobarbitone, Sodium Valproate, Ethosuximide, and Sulthiame in the Rat

The effect of prolonged treatment (10 days) with the anticonvulsant drugs diphenylhydantoin (DPH), phenobarbitone, sodium valproate, ethosuximide and sulthiame, both singly and in combination, on regional rat brain amino acid neurotransmitter concentrations (GABA, glutamate, aspartate and taurine) were assessed. DPH had a major effect in the cerebellum and hypothalamus in that it significantly reduced cerebellar GABA, taurine and aspartate and hypothalamic GABA and aspartate. Sodium valproate significantly elevated GABA and taurine in most regions. Aspartate and glutamate were less affected. Phenobarbitone significantly elevated GABA concentrations in all brain regions, while taurine concentration was only elevated in the cerebral cortex. Ethosuximide induced changes were small compared to the other anticonvulsants while sulthiame produced complex changes. Anticonvulsant drugs administered in combination resulted in complex changes, suggesting that their mode of action is different.     

Uptake of 36Cl and 22Na by the Choroid Plexus-Cerebrospinal Fluid System: Evidence for Active Chloride Transport by the Choroidal Epithelium

Abstract: Cl and Na transport by the lateral ventricle (LVCP) and fourth ventricle (4VCP) choroid plexuses were examined by kinetic analysis of ³⁶Cl and ²²Na uptake into the choroid plexus-CSF system of the adult rat. Both radioisotopes required more than 5 h to reach steady-state distribution in the in vivo choroid plexuses and CSF after intraperitoneal injection. Whereas the LVCP and 4VCP ³⁶Cl steady-state spaces were comparable (55–56%), the 4VCP ²²Na space (39%) tended to be greater than the LVCP ²²Na space (36%). No evidence for inexchangeable Cl or Na was found for the choroid plexuses; the radioisotopic and chemical spaces were not significantly different. Choroid plexus ³⁶Cl and ²²Na uptake curves were resolved into two components, a fast component ( t _1/2 0.02–0.05 h) and a slow component ( t _1/2 0.85–1.93 h). By analysis of the distribution of [³H]inulin, [³H]mannitol, and ⁵¹Cr-tagged erythrocytes within the choroid plexuses, the fast component of ³⁶Cl and ²²Na uptake was found to represent extracellular and erythrocyte contributions to the tissue radioactivity, whereas the slow component represented isotope movement into the epithelial cell compartment. The calculated cell [Cl] of LVCP and 4VCP, 67 mmol/kg cell water, was 3.9 times greater than that predicted by the membrane potential for passive distribution. It is postulated that Cl is actively transported into the choroid epithelial cell across the basolateral membrane; the energy source for active Cl transport may be the Na electrochemical potential gradient (˜90 mV), which is twice that of the Cl electrochemical potential gradient (˜45 mV).     

A 31P-NMR study of the cross-membrane pH gradient induced by ATP hydrolysis in mitochondria

³¹P-NMR has been used to study the increase of ΔpH in mitochondria by externally added ATP. Freshly prepared mitochondria was treated with N-ethylmaleimide to inhibit the exchange between internal and external P_i. Upon addition of ATP, phosphocreatine (30 mM) and creatine kinase to a NMR sample of mitochondria suspension (approx. 120 mg protein/ml) at 0°C, an increase of ΔpH by approx. 0.5 pH unit was observed. However the increased ΔpH could not be maintained, but slowly decayed along with the increase of external ADP/ATP ratio. Further addition of valinomycin to the suspension induced a larger ΔpH (approx. 1) which was maintained by the increased rate of internal ATP hydrolysis as seen in the growth of the internal P_i peak intensity in NMR spectra and the concomitant decrease of the external phosphocreatine peak. The external P_i and ATP peaks stayed virtually constant. When carboxyatractyloside was added to inhibit the ATP/ADP translocase, the internal P_i increase was stopped and the ΔpH decayed. These observations in conjunction with those made earlier in respiring mitochondria clearly show the reversible nature of the ATPase function in which the internal ATP hydrolysis is associated with outward pumping of protons.