Galleria mellonella nerve cords in vitro: stage-specific survival and differential responsiveness to beta-ecdysone

The survival of ventral nerve cord segments of Galleria mellonella in tissue culture medium, ascertained by histological and biochemical criteria, ability to shorten when transplanted, and responsiveness to β-ecdysone, is correlated with the stage of development of the donor, and with the presence of a connective tissue sheath, the neural lamella, about the segment. After only 18 hr in vitro connectives which have lost the neural lamella during metamorphosis no longer have the capacity to shorten when transplanted or when exposed to β-ecdysone in the culture medium. By contrast, after 7 days in vitro connectives with a neural lamella shorten appreciably when β-ecdysone is added, or when they are exposed to the humoral milieu of a host undergoing metamorphosis. β-Ecdysone stimulates the incorporation of uridine-5-³H in segments both with and without the neural lamella, but only in segments which have previously begun their metamorphosis. Since shortening in response to β-ecdysone occurs only in connectives which have already begun to shorten, β-ecdysone appears to accelerate physiological processes underway before it is added to the medium rather than initiate metamorphosis in cultured nerve cords.     

O-Glycosidically linked oligosaccharides from peptidorhamnomannans ofSporothrix schenckii

-Elimination of peptidorhamnomannans purified from yeast-like and mycelial phases ofSporothrix schenckii released neutral and acidic reduced oligosaccharides that were O linked to serine and/or threonine. Man-(1–2)Man-ol, Rha(1–3)Man(1–2)Man-ol, Rha(1–4)GlcA(1–2)Man(1–2)Man-ol, and Rha(1–4)[Rha(1–2)] GlcA(1–2)Man(1–2)Man-ol were characterized based on methylation analysis, proton magnetic resonance and fast atom bombardment mass spectrometry.Abbreviations FAB fast atom bombardment - GLC gas liquid chromatography - GlcA d-glucopyranosyluronic acid - Man d-mannopyranose - Man-ol d-mannitol - MS mass spectrometry - NMR nuclear magnetic resonance - Rha l-rhamnopyranose     

Glycolate photoproduction by free and alginate-entrapped cells of Chlamydomonas reinhardtii

Summary Chlamydomonas reinhardtii cells provide an effective system for glycolate photoproduction, operative during 30 h when they are growing under low CO₂, in the presence of 1 mM aminooxyacetate and 50 M acetazolamide. Glycolate excretion by the cells can proceed for about 4 days if every other 12 h a high CO₂ level is restored in the culture in the absence of inhibitors. The immobilized system in alginate beads has about a twofold higher glycolate photoproduction rate (23 mol·mg chlorophyll (Chl)^–1·h^–1) than free-living cells (12 mol · mg Chl^–1 · h^–1). Offprint requests to: C. Vílchez     

Voltage-gated chloride currents in cultured canine tracheal epithelial cells

Summary Chloride ions (Cl^–) are concentrated in airway epithelial cells and subsequently secreted into the tracheal lumen by downhill flux through apical Cl^– channels. We have studied Cl^– currents in cultured canine tracheal cells using the whole-cell voltage-clamp technique. Ultrastructural techniques demonstrated that the cells used in the electrophysiological experiments possessed apical membrane specializations known to be present in the intact, transporting cell type. Cultured cells 2–6 days old were characterized by an input resistance of 3.4±0.8 G (n=11) and a capacitance of 63.8±10.8 pF (n=26). A comparison of 3 and 4 day-old cells with 5 and 6 day-old cells showed that the input resistance decreased almost 50%, and the cell capacitance and the inward and outward currents increased concomitantly approximately 200%. Cultured cells 3–4 days old held at –40 mV produced currents of 196±22 pA at 50 mV and –246±27 pA at –90 mV (n=212) with pipette and bath solutions containing primarily 140 KCl and 140 NaCl, respectively. The chloride channel blocker diphenylamine-2-carboxylate (DPC, 100 m) suppressed whole-cell currents by 76.8% at 60 mV; however, currents were unaffected by the stilbenes SITS (1mm) and DNDS (1–30 m). Replacement of K⁺ with Cs⁺ in the pipette solution did not affect the outward current, the current reversal potential, or the input resistance of the cells, indicating that the current was not significantly K⁺ dependent when the intrapipette solution was buffered to a Ca²⁺ concentration of 20nm. The Cl^–/Na⁺ permeability ratio was estimated to be greater than 11 as calculated from reversal potential measurements in the presence of an internal to external NaCl concentration ratio of 12. Current equilibrium permeabilities, relative to Cl^– were: I^– (2.9)NO ₃ ^– (1.1)Br^– (1.1)Cl^– (1.0)F^– (0.93)MeSO ₄ ^– (0.19)gluconate (0.18)aspartate (0.14). Depolarizations to potentials greater than 20 mV elicited a time-dependent component in the outward current in 71% of the cells studied. Currents inactivated with a double exponential time course at the most depolarized voltages. Recovery from inactivation was fast, holding potential-dependent, and followed a double exponential time course. Current amplitude was increased via a cAMP-dependent pathway as has been demonstrated for single Cl-selective channels in cell-attached patches from cultured canine and human tracheal epithelial cells. Forskolin, an activator of adenylate cyclase, produced a 260% increase in the outward current at +50 mV. In summary, cultured canine tracheal cells have a single resting conductance that is Cl^– selective, voltage-dependent, and modulated by a cAMP-dependent mechanism. This preparation appears to be appropriate for analysis of cellular modulation of airway Cl^– channels and Cl^– secretion.     

The nature of the neutral Na+−Cl−-coupled entry at the apical membrane of rabbit gallbladder epithelium: II. Na+−Cl− symport is independent of K+

Summary In the epithelium of rabbit gallbladder, in the nominal absence of bicarbonate, intracellular Cl^– activity is about 25mm, about 4 times higher than intracellular Cl^– activity at the electrochemical equilibrium. It is essentially not affected by 10^–4 m acetazolamide and 10^–4 m 4-acetamido-4-isothiocyanostilbene-2,2-disulfonate (SITS) even during prolonged exposures; it falls to the equilibrium value by removal of Na⁺ from the lumen without significant changes of the apical membrane potential difference. Both intracellular Cl^– and Na⁺ activities are decreased by luminal treatment with 25mm SCN^–; the initial rates of change are not significantly different. In addition, the initial rates of change of intracellular Cl^– activity are not significantly different upon Na⁺ or Cl^– entry block by the appropriate reduction of the concentration of either ion in the luminal solution. Luminal K⁺ removal or 10^–5 m bumetanide do not affect intracellular Cl^– and Na⁺ activities or Cl^– influx through the apical membrane. It is concluded that in the absence of bicarbonate NaCl entry is entirely due to a Na⁺–Cl^– symport on a single carrier which, at least under the conditions tested, does not cotransport K⁺.     

Hämatologische Beobachtungen an Regenbogenforellen (Salmo gairdneri RICH.). V. Die Erythozytenverteilungskurve

Zusammenfassung Für die Fischpathologie wird als differentialdiagnostisches Verfahren die Erythozytenmessung vorgeschlagen. Zur Darstellung der Erythrozytenverteilungskurve wird wegen der elliptischen Form der Fisch-Erythrozyten die in der Humanmedizin übliche Technik modifiziert.An 102 Regenbogenforellen (Salmo gairdneri) beiderlei Geschlechts im Alter von 1 bis 3 Jahren wurde versucht, die Normalkurve für gesunde Tiere dieser Population zu ermitteln. Die Größenverteilung der Erythrozyten ergibt eine Gauss-Glockenkurve, deren Gipfel in die Größenklasse 30–35 µm²/ fällt, und deren Basis von 15–20 µm²/ bis 55–60 µm²/ reicht.Zwischen den Price-Jones-Kurven der Milchner (52 Individuen) und Rogner (50 Individuen) bestehen keine signifikanten Unterschiede. Ebenso konnte keine Beeinflussung dieser Normalkurve durch unterschiedliches Alter oder jahreszeitliche Einflüsse — untersucht wurden in dieser Hinsicht die F₂ — beobachtet werden.

---

Summary As differential-diagnostic feature in the fish pathology it is suggested to measure the red blood cells. The normal technique used in human medicine for plotting the distribution curve of the red blood cells is modified, because the red blood cells of the fish are of elliptical shape.102 rainbow trouts (Salmo gairdneri), both, male and female, and 1–3 years of age, were examined to find the normal curve for healthy animals of this population. The size distribution of the red blood cells show a bell-shaped Gauss-curve, the peak of which is 30–35 µm²/, and the basis of which reaches from 15–20 µm²/ to 55–60 µm²/.The Price-Jones-curves show no significant differences between the milters (52 individuals) and the spawners (50 individuals). Besides, changes caused by different age or seasonal influences could not be observed on this normal curve. Analyzed were the F₂ individuals.

---

---

Institut für Siedlungswasserbau und Wassergütewirtschaft der Universität Stuttgart Biologische Abteilung - Arbeitsgruppe Haider     

Effects of local anesthetics on phospholipid topology and dopamine uptake and release in rat brain synaptosomes

Summary The effects of local anesthetics on the topology of aminophospholipids and on the release and uptake of dopamine in rat brain synaptosomes have been examined. A metabolically intact preparation of synaptosomes was prepared which maintains aminophospholipid asymmetry and the capacity for sodium-driven uptake and depolarization-dependent release of dopamine. Incubation of synaptosomes with local anesthetics at 37°C induced perturbations in the topology of aminophospholipids as determined by their reactivities to the covalent probe trinitrobenzenesulfonic acid. The reaction of trinitrobenzenesulfonate with phosphatidylethanolamine and phosphatidylserine was inhibited 10–20% by low concentrations of tetracaine (1–100 m) and enhanced by high concentrations (0.3–1.0mm). Other local anesthetics showed a similar biphasic effect with a potency order of dibucaine>tetracaine>lidocaineprocaine. K⁺-stimulated, Ca²⁺-dependent release of [³H]dopamine was inhibited significantly at low concentrations of tetracaine (1–10 m) but enhanced at higher concentrations (0.1–1.0mm). Dibucaine and procaine had a similar biphasic effect on the dopamine release. For each of the local anesthetics tested, the inhibition of the reaction of phosphatidylethanolamine and phosphatidylserine with trinitrobenzenesulfonate occurred at concentrations which were shown also to inhibit the release of [³H]dopamine. Local anesthetics were shown to inhibit uptake of [³H]dopamine with a potency order which reflects their potency in producing anesthesia. The inhibition of dopamine uptake by dibucaine, tetracaine, lidocaine, or procaine was characterized by inhibitory constants (K _I ) of 1.8±0.4 m, 27±5 m, 190 m and 0.5mm, respectively.Abbreviations TNBS 2,4,6-trinitrobenzene sulfonate - PE phosphatidylethanolamine - PS phosphatidylserine - ESR electron spin resonance - TLC thin-layer chromatography - DA dopamine     

Synapses in the cerebral ganglion of adult Ciona intestinalis

Summary Electron microscopy of the synaptic morphology of synapses in the cerebral ganglion of the adult ascidian (sea squirt) Ciona intestinalis reveals that the synapses are restricted to the central neuropil of the ganglion. Many of the synapses show a polarity of structure such that pre and post synaptic parts can be identified. The vesicles in the presynaptic bag are of two main diameters 80 and 30 nm respectively. The large vesicles have electron dense contents that vary both in their capacity and dimensions.The pre and postsynaptic membranes are more electron dense than the surrounding membranes, but they are only slightly thicker. Both the pre and post synaptic membranes have electron dense dots some 10 nm in diameter associated with their cytoplasmic surfaces. Sometimes the presynaptic membrane has larger peg-like projections between the vesicles. Associated with the post synaptic membrane are tubules some 10 nm in diameter. These tubules may be the dots cut obliquely.The synaptic cleft material is more electron dense than the surrounding intercellular material, and in it there is a dense line made up of granules about 3–5 nm in diameter. This dense line is usually mid way between the pre and post synaptic membranes, but may be nearer the postsynaptic membrane.No tight junctions between adjacent nerve process profiles have been observed.I wish to thank Professors J. Z. Young, F. R. S. and E. G. Gray for much advice and encouragement, also Dr. R. Bellairs for the use of electron microscope facilities and Mr. R. Moss and Mrs. J. Hamilton for skillful technical assistance.     

Interactions between anion exchange and other membrane proteins in rabbit kidney medullary collecting duct cells

Summary In separated outer medullary collecting duct (MCD) cells, the time course of binding of the fluorescent stilbene anion exchange inhibitor, DBDS (4,4-dibenzamido-2,2-stilbene disulfonate), to the MCD cell analog of band 3, the red blood cell (rbc) anion exchange protein, can be measured by the stopped-flow method and the reaction time constant, _DBDS, can be used to report on the conformational state of the band 3 analog. In order to validate the method we have now shown that the ID_50,DBDS,MCD (0.5±0.1 m) for the H₂-DIDS (4,4-diisothiocyano-2,2-dihydrostilbene disulfonate) inhibition of _DBDS is in agreement with the ID_50,Cl ^–,_MCD (0.94±0.07 m) for H₂-DIDS inhibition of MCD cell Cl^– flux, thus relating _DBDS directly to anion exchange. The specific cardiac glycoside cation transport inhibitor, ouabain, not only modulates DBDS binding kinetics, but also increases the time constant for Cl^– exchange by a factor of two, from _Cl=0.30±0.02 sec to 0.56±0.06 sec (30mm NaHCO₃). The ID_50,DBDS,MCD for the ouabain effect on DBDS binding kinetics is 0.003±0.001 m, so that binding is about an order of magnitude tighter than that for inhibition of rbc K⁺ flux (K _I,K ⁺,_rbc=0.017 m). These experiments indicate that the Na⁺,K^–-ATPase, required to maintain cation gradients across the MCD cell membrane, is close enough to the band 3 analog that conformational information can be exchanged. Cytochalasin E (CE), which binds to the spectrin/actin complex in rbc and other cells, modulates DBDS binding kinetics with a physiological ID_50,DBDS,MCD (0.076±0.005 m); 2 m CE also more than doubles the Cl^– exchange time constant from 0.20±0.04 sec to 0.50±0.08 sec (30mm NaHCO₃). These experiments indicate that conformational information can also be exchanged between the MCD cell band 3 analog and the MCD cell cytoskeleton.     

Inhibition of tumor growth in mice treated with synthetic muramyl dipeptide

Summary Treatment with synthetic MDP inhibited growth of transplantable, chemically induced tumors in syngeneic mice. The tumor-inhibitory effect was dependent on the schedule of MDP administration.Growth of SC transplants of a nonmetastasizing, MC-induced fibrosarcoma, MC11, was inhibited by local treatment with 200 g and 1,000 g MDP given SC 5–7 weeks before challenge. Treatment with lower (10 g and 100 g) doses of MDP and shorter (1–4 weeks) time intervals was not effective. Single doses of MDP (10–1,000 g) 1–3 weeks after challenge had no effect.Growth of IV-inoculated, metastasizing AAT-induced hepatoma A was inhibited by IV injections of 20 g MDP given 1 and 2 days prior to the challenge. Significant increases in the survival of hepatoma-bearing mice were observed only after injections of MDP incorporated in multilamellar liposomes.Abbreviations MDP n-acetylmuramyl-l-alanyl-d-isoglutamine - B10 C57BL/10ScSnPh mice - MC 3-methylcholanthrene - ATT o-amino-azotoluene - PBS phosphate-buffered saline     

Transport of potassium inChara australis: II. Kinetics of a symport with sodium

Summary An electrogenic K⁺–Na⁺ symport with a high affinity for K⁺ has been found inChara (Smith & Walker, 1989). Under voltage-clamp conditions, the symport shows up as a change in membrane current upon adding either K⁺ or Na⁺ to the bathing medium in the presence of the other. Estimation of kinetic parameters for this transport has been difficult when using intact cells, since K⁺–Na⁺ current changes show a rapid falling off with time at K⁺ concentrations above 50 m. Cytoplasm-enriched cell fragments are used to overcome this difficulty since they do not show the rapid falling off of current change seen with intact cells. Current-voltage curves for the membrane in the absence or presence of either K⁺ or Na⁺ are obtained, yielding difference current-voltage curves which isolate the symport currents from other transport processes. The kinetic parameters describing this transport are found to be voltage dependent, withK _m for K⁺ ranging from 30 down to 2 m as membrane potential varies from –140 to –400 mV, andK _m for Na⁺ ranging between 470 and 700 m over a membrane potential range of –140 to –310 mV.Two different models for this transport system have been investigated. One of these involves the simultaneous transport of both the driver and substrate ions across the membrane, while the other allows for the possibility of the two ions being transported consecutively in two distinct reaction steps. The experimental results are shown to be consistent with either of these cotransport models, but they do suggest that binding of K⁺ occurs before that of Na⁺, and that movement of charge across the membrane (the voltage-dependent step) occurs when the transport protein has neither K⁺ nor Na⁺ bound to it.     

Partial purification and characterization of β-hydroxybutyric acid dehydrogenase of a methylotrophic bacterium,Pseudomonas 135

Summary An intracellular enzyme, d(—)--hydroxybutyric acid dehydrogenase involved in an intracellular poly-d(—)--hydroxybutyric acid degredation was isolated from a facultative methylotrophic bacterium, Pseudomonas 135, grown on methanol as a sole carbon and energy source. This enzyme was partially purified to 11.6-fold by ammonium sulphate fractionation and a dye-affinity chromatography. The enzyme catalysed simultaneously the oxidation of d(—)--hydroxybutyric acid (D-HB) and the reduction of acetoacetate. The optimum pH was 8.5 for the oxidation reaction and 5.5–6.0 for the reduction reaction, and the enzyme was stable for 2 weeks at — 20° C. The K _m values for oxidation and reduction reactions were determined as 1.84 mm for D-HB, 0.244 mm for NAD⁺, 0.319 mm for acetoacetate and 0.032 mm for NADH, respectively. It was also found that d-lactate and NADH significantly inhibited the oxidation reaction by competitive inhibition, and acetoacetate by non-competitive inhibition, respectively. The inhibition constants were determined as 1.49 mm for d-lactate, 0.196 mm for NADH and 1.82 mm for acetoacetate, respectively. According to an experiment with resting cells, it seemed that the enzyme was constitutive. Correspondence to: J. M. Lebeault     

Physiological and anatomical properties of Leydig cells in the segmental nervous system of the leech

Summary Each of the 21 segmental ganglia in the American leechMacrobdella decora contains a pair of Leydig cells (ca. 45 m) each of which is located in a posteriolateral glial packet. Leydig cells exhibit spontaneous action potentials (1–10/s) whose duration and undershoot depend upon membrane polarization. The two Leydig cells within each ganglion are bidirectionally-coupled (V ₂/V ₁0.3). Pairs of ipsilateral Leydig cells within adjacent ganglia are mutually excitatory such that an impulse in one generates an impulse in the other. The interganglionic latency for any cell pair is constant regardless of the direction of impulse conduction and is unchanged by 20 mM Mg²⁺ saline. These data indicate that the interactions are not mediated by chemical synapses. Additionally, the results of collision experiments lead us to infer that ipsilateral Leydig cell pairs utilize common axonal pathways for interganglionic interactions. If Leydig cells are driven by current injection to fire impulses at frequencies of six to ten per s, cells in adjacent ganglia exhibit impulse failures. The combination of spontaneous activity, intraganglionic coupling and interganglionic interactions results in the generation of constant, low frequency impulse activity and can cause impulse reverberations.The branching pattern of Leydig cells filled with HRP is consistent with their functional properties and connectivity. Each cell sends axons to both adjacent ganglia through the ipsilateral connectives and projects to the periphery only by the lateral roots of these adjacent ganglia. This unusual morphology was verified Lucifer Yellow CH.In addition to intraganglionic dye-coupling, dye coupling was occasionally evident between ipsilateral cells in adjacent ganglia. Electron microscopy of Leydig cells depicts abundant 100 nm granules in both their somata and neuropilar processes. Although this fine structure suggests a neurosecretory role, we were unable to discern a peripheral function for these neurons.Abbreviation H R P horseradish peroxidase     

Effects of monensin on ATP levels and cell functions in rat liver and lung in vitro

Summary Effects of the proton-alkali cation-exchanging ionophore, monensin, on aspects of cellular metabolism and ionic exchanges have been studied in rat tissues in vitro. Incubation of liver slices at 38°C with 0.1 m monensin induced timedependent vesiculation, initially in the Golgi region, reduction of ATP content and of protein synthesis. At 1 m, monensin also reduced net, active movements of K⁺, Na⁺, Cl^– and water in liver slices and inhibited state 3 respiration in isolated mitochondria. The respiratory inhibitor, amytal, similarly reduced ATP content and protein synthesis at concentrations lower than those inhibiting ion transport in slices. Low concentrations of monensin (0.1–1.0 m) had similar effects on ATP and ion transport in slices of adult lung. By contrast, late-fetal liver and lung were much less sensitive to monensin; in these tissues, glycolysis sustained substantial levels of ATP. Monensin also induced vesiculation of the Golgi apparatus in fetal lung cells. It is concluded that by lowering ATP levels, monensin can markedly alter various metabolic activities in those cells which depend primarily on oxidative phosphorylation for their metabolic energy.     

Particles resembling microbodies in normal and neoplastic perianal glands of dogs

Summary By electron microscopy, the parenchymal cells of the perianal glands of dogs contain granules which have the morphological features of microbodies (peroxisomes) including marginal plates and, occasionally, dense nucleoids. Like microbodies, they are occasionally attached to the endoplasmic reticulum. Histochemical evidence is presented suggesting that they contain at least one of the peroxisomal enzymes, L--hydroxy acid oxidase. The granules of a perianal gland adenoma showed abnormal morphologic variations.Mrs. Murtie Still, Mrs. Bertha McClure and Mr. Bob White gave valuable technical assistance.     

Specific binding of kainic acid to purified subcellular fractions from rat brain

The subcellular distribution of kainic acid (KA) binding sites in rat brain has been studied using a microcentrifugation assay. KA did not bind to myelin or brain cytosol and had few or no binding sites in the nuclear fraction. However, it bound to microsomal components (K _d =128–136 nM; 2.5–4.8 pmol/mg protein), purified synaptic plasma membranes (SPM) (K _d =45–71 nM; 5.8–6.5 pmol/mg), and purified cell-body and intraterminal mitochondria (K _d =11–31 nM; 0.4–1.1 pmol/mg). Bound KA could be totally displaced byl-glutamate orl-aspartate, but several putative antagonists of these amino acids (nuciferin, compound HA-966, 2-amino-4-phosphonobutyrate, and 2-amino-3-phosphonoproprionate) failed to displace KA or did so at very high concentrations (4 mM). Glutamic acid diethyl ester (GDEE) andd,l--aminoadipate (-AA) were more effective (IC₅₀, 0.2–0.8 mM) and showed differential effects in their capacity to displace KA bound to the various subcellular fractions. Thus, GDEE only displaced 40–60% of the KA bound by SPM or mitochondria and did not prevent the binding of KA to microsomes. -AA, on the other hand, was more effective in preventing the binding of KA at high concentrations and displaced between 80 and 100% of the drug. Both compounds showed biphasic curves of KA displacement from synaptic plasma membranes and mitochondria. The overall results indicate the presence of multiple binding sites for KA in brain cells and suggest that KA does not act exclusively at synaptic glutamate receptors. The mechanism of KA action is most likely quite complex, and the drug probably acts at multiple binding sites affecting a number of processes.     

The effect of poly-l-lysine,amiloride and methyl-l-lysine on gill ion transport and permeability in the rainbow trout

Summary The action of poly-l-lysine (PLL) on Na and Cl transport across freshwater fish gills was studied. Low concentrations (10^–6M) were added to the external medium for brief periods (1–5 min), then removed. During the next 20 min there was a rapid net loss of Na (117±17 Equiv[100 g]^–1 hr^–1) and Cl (129±17 Equiv[100 g]^–1 hr^–1). Both values are an order of magnitude larger than unidirectional effluxes in control fish. The efflux of both ions decreased to control values within 60 min after application and removal of PLL. In contrast, unidirectional influxes (J _in ^Cl andJ _in ^Na ) were inhibited by about 40% and showed no sign of returning to the original rates for 3 hr. Thus, PLL has two independent actions, causing a large increase in gill permeability which is reversible within an hour and a partial inhibition of influx which showed no sign of reversing for 2–3 hr. When PLL was applied for a longer period (60 min, the results were qualitatively similar but the permeability change was larger and persisted longer. These effects were compared with those of the small organic amines, amiloride and methyl-l-lysine. The latter inhibitedJ _in ^Na , but there was no other similarity to PLL. Neither affected sodium efflux, nor did they have any effect on Cl movements, in or out, across the gill. Inhibition ofJ _in ^Na , was rapidly and completely reversible, amd amiloride was shown to act by competing with Na for an entry site.     

Electrogenic and electroneutral transport modes of renal Na/K ATPase reconstituted into proteoliposomes

Summary This paper describes measurements of electrical potentials generated by renal Na/K-ATPase reconstituted into proteoliposomes, utilizing the anionic dye, oxonol VI. Calibration of absorption changes with imposed diffusion potentials allows estimation of absolute values of electrogenic potentials.ATP-dependent Na_cyt/K_exc exchange in K-loaded vesicles generates large potentials, up to 250 mV. By comparing initial rates or steady-state potentials with ATP-dependent²²Na fluxes in different conditions, it is possible to infer whether coupling ratios are constant or variable. For concentrations of Na_cyt (2–50mm) and ATP (1–1000 m) and pH's (6.5–8.5), the classical 3Na_cyt/2K_exc coupling ratio is maintained. However, at low Na_cyt concentrations (<0.8mm), the coupling ratio is apparently less than 3Na_cyt/2K_exc.ATP-dependent Na_cyt/congener_exc exchange in vesicles loaded with Rb, Cs, Li and Na is electrogenic. In this mode congeners, including Na_exc, act as K_exc surrogates in an electrogenic 3Na_cyt/2congener_exc exchange. (ATP+Pi)-dependent K_cyt/K_exc exchange in K-loaded vesicles is electroneutral.ATP-dependent uncoupled Na flux into Na- and K-free vesicles is electroneutral at pH 6.5–7.0 but becomes progressively electrogenic as the pH is raised to 8.5. The²²Na flux shows no anion specificity. We propose that uncoupled Na flux is an electroneutral 3Na_cyt/3H_exc exchange at pH 6.5–7.0 but at higher pH's the coupling ratio changes progressively, reaching 3Na/no ions at pH 8.5. Slow passive pump-mediated net K uptake into Na- and K-free vesicles is electroneutral, and may also involve K_cyt/H_exc exchange.We propose the general hypothesis that coupling ratios are fixed when cation transport sites are saturated, but at low concentrations of transported cations, e.g., Na_cyt in Na/K exchange and H_exc in uncoupled Na flux, coupling ratios may change.     

A quantitative study of synaptic interconnections in the dorsal lateral geniculate nucleus of the cat

Summary 2,700 synaptic contacts have been classified according to criteria described in an accompanying paper and the results summarized in tabular form. Only about 20% of the synaptic contacts in laminae A and A1 are formed by axons identifiable as retinogeniculate fibers. About 1/4 of these retinogeniculate synapses are axo-axonal. Approximately 45% of the contacts in these laminae are formed by axons tentatively identifiable as corticogeniculate fibers; about 35% by presumed intrageniculate fibers. Close to one half of the synapses occur in encapsulated synaptic zones, where grapelike dendritic appendages are related mainly to intrageniculate and retinogeniculate axons, and about half lie in interstitial zones, where corticogeniculate and some intrageniculate axons contact distal dendritic segments.Regions of the nucleus receiving from peripheral parts of the retina have relatively more corticogeniculate synapses, and have fewer intrageniculate synapses in the encapsulated zones than do regions receiving from the central parts of the retina.Most of the tissue in lamina B resembles the interstitial zones of laminae A and A1 and over 2/3 of the contacts in lamina B may prove to be corticogeniculate. The retinogeniculate fibers in this lamina are associated with relatively few other axons in simple, small encapsulated zones.Supported by Grant NB 06662 from the USPHS. The skillful technical assistance given by Mrs. E. Langer during the course of this work is gratefully acknowledged.     

The nature of the neutral Na+−Cl− coupled entry at the apical membrane of rabbit gallbladder epithelium: III. Analysis of transports on membrane vesicles

Summary In rabbit gallbladder epithelium, a Na⁺/H⁺, Cl^–/HCO ₃ ^– double exchange and a Na⁺–Cl^– symport are both present, but experiments on intact tissue cannot resolve whether the two transport systems operate simultaneously. Thus, isolated apical plasma membrane vesicles were prepared. After preloading with Na⁺, injection into a sodium-free medium caused a stable intravesicular acidification (monitored with the acridine orange fluorescence quenching method) that was reversed by Na⁺ addition to the external solution. Although to a lesser extent, acidification took place also in experiments with an electric potential difference (PD) equal to 0. If a preset pH difference (pH) was imposed ([H⁺]_in>[H⁺]_out, PD=0), the addition of Na-gluconate to the external solution caused pH dissipation at a rate that followed saturation kinetics. Amiloride (10^–4 m) reduced the pH dissipation rate. Taken together, these data indicate the presence of Na⁺ and H⁺ conductances in addition to an amiloride-sensitive, electroneutral Na⁺/H⁺ exchange.An inwardly directed [Cl^–] gradient (PD=0) did not induce intravesicular acidification. Therefore, in this preparation, there was no evidence for the presence of a Cl^–/OH^– exchange.When both [Na⁺] and [Cl^–] gradients (outwardly directed, PD=0) were present, fluorescence quenching reached a maximum 20–30 sec after vesicle injection and then quickly decreased. The decrease was not observed in the presence of a [Na⁺] gradient alone or the same [Na⁺] gradient with Cl^– at equal concentrations at both sides. Similarly, the decrease was abolished in the presence of both Na⁺ and Cl^– concentration gradients and hydrochlorothiazide (5×10^–4 m). The decrease was not influenced by an inhibitor of Cl^–/OH^– exchange (10^–4 m furosemide) or of Na⁺–K⁺–2Cl^– symport (10^–5 m bumetanide).We conclude that a Na⁺/H⁺ exchange and a Na⁺–Cl^– symport are present and act simultaneously. This suggests that in intact tissue the Na⁺–Cl^– symport is also likely to work in parallel with the Na⁺/H⁺ exchange and does not represent an induced homeostatic reaction of the epithelium when Na⁺/H⁺ exchange is inhibited.