Growth factors modulate junctional cell-to-cell communication

Summary The epidermal growth factor (EGF) and the platelet-derived growth factor (PDGF) inhibit gap junctional communication in the mammalian cell lines NRK and BalbC 3T3: cell-to-cell transfer of a 400-dalton tracer molecule is reduced and junctional conductance is reduced. The inhibition of cell-to-cell transfer is reversible and dose dependent; half-maximal effects are obtained at 10^–9 and 10^–11 m concentrations of EGF and PDGF, respectively. The response of junctional conductance is detectable within 2 min of EGF application and reaches a maximum within 10 min. It is among the earliest cellular responses to this growth factor and may be significant in the regulation of growth. The response is lacking in EGF receptor-deficient NIH 3T3 cells. The transforming factor (TGF) enhances junctional communication in BalbC 3T3: cell-to-cell transfer is increased over a period of 8 hr. But in NRK cells, where it upregulates EGF receptors, TGF reduces junctional communication synergistically with EGF.     

Interrelationship of nitrogen fixation,hydrogen evolution and photoreduction in Rhodospirillum rubrum

Summary Rhodospirillum rubrum was grown: 1. photoheterotrophically on a medium containing dl-malate as the carbon source and ammonium chloride as the nitrogen source (medium No. 1); 2. phototrophically with N₂ and dl-malate (medium No. 2); 3. photoautotrophically with N₂, CO₂ and H₂ (medium No. 3).Resting cells derived from these cultures were tested for their ability to photoreduce CO₂, evolve H₂ and fix N₂. Only cells which were grown in medium No. 2 were able to perform all three gas exchanges. The activity pattern of gas exchanges altered in a characteristic way during the growth cycle of the bacterial culture. Cells newly transferred to medium No. 2 showed an enormous increase in the rate of H₂ evolution, which dropped sharply when all l-malate had been used up. The rate of photoreduction of CO₂ increased steadily and reached a maximum level after 120 h. The nitrogen fixing activity remained constant during the whole growth cycle.The yields of H₂ produced per mole of l-malate added were measured as a function of cell age. Only very young cultures gave appreciable yields, which dropped gradually with increasing age.The function of the carbon source is discussed as a regulating factor for photoreduction and hydrogen evolution.     

TES and HEPES buffers in mammalian cell cultures and viral studies: Problem of carbon dioxide requirement

In order to evaluate TES and HEPES as a buffer system for cell culture, the proliferative capacities of cells of several mammalian cell lines in the medium buffered with either of these compounds were examined in cultures in stoppered and open flasks at high and low cell densities. When cultivated in stoppered flasks, cells grew equally well or even better in TES- and HEPES-buffered medium than in NaHCO₃-buffered medium irrespective of cell culture density. In open flasks or Petri dishes in TES- or HEPES-buffered medium, however, the proliferative capacity of cells in low density cultures was limited. The inhibition of cell growth in the latter condition was restored (1) as the cell density of the cultures increased; (2) by feeding continuously the cultures with the gas produced by high density cultures; (3) by introducing a small amount of CO₂ to the environment.These and other evidences presented suggest that, in agreement with the prevailing notions, CO₂ is required by cells as an essential nutrient for growth, and that the desired level of CO₂ in culture can be maintained efficiently by its production by even a small number of cells in culture as long as the culture flasks are stoppered. If flasks are not stoppered, however, the level of CO₂ tension is determined by an equilibrium between the rate of its production by the cells and that of escape from culture to air, resulting in the observed failure in growth of cells in TES- and HEPES-buffered medium at low cell densities unless cultures were further supplemented with added CO₂.     

Diacylglycerol downregulates junctional membrane permeability. TMB-8 blocks this effect

Summary We tested the question whether junctional cell-to-cell communication is regulated by the diacylglycerol branch of the phosphoinositide transmembrane signal pathway. Cultured epithelial rat liver cells were treated with the synthetic diacylglycerol 1-oleoyl-2-acetyl glycerol, while their junctional permeability was probed with the microinjected 443-dalton fluorescent tracer Lucifer Yellow. The treatment reduced junctional permeability (without affecting Lucifer permeability of nonjunctional cell membrane). The effect was dose dependent, with a threshold of about 25 g diacylglycerol/ml in sparse cultures and about 50 g/ml in confluent cultures. The reduction of junctional permeability began within 3 min of diacylglycerol application, peaked within 20 min, and reversed spontaneously within 90 min. The phorbol ester TPA mimicked the diacylglycerol effect, but the (spontaneous) reversal was slower. We propose that cell-to-cell communication is under dual physiological control: an upregulatory one, as exerted by the cyclic AMP signal route (Loewenstein, W.R., 1985,Biochem. Soc. Symp. London,50: 43–58), and a downregulatory one, by the diacylglycerol signal route.TMB-8 (54–70 m)—a blocker of intracellular Ca²⁺ mobilization-impeded the diacylglycerol action on junctional permeability. It prevented the effect of low diacylglycerol doses completely and it markedly reduced the effect of high doses. (It also counteracted the effect of TPA.) Ca²⁺ thus emerges as a possible candidate for a role in the junctional downregulation by the diacylglycerol signal route. We tentatively advance two models. In one, leaning closely on the Calcium Hypothesis of cell-to-cell channel regulation (Loewenstein, W.R., 1966,Ann. N.Y. Acad. Sci. 137:441–472), Ca²⁺ mediates the action of the route on the channel. In the other, Ca²⁺ acts farther removed from the channel, on protein kinase C.Calmidazolium (5–10 m)—an inhibitor of calmodulin-activated proteins—did not prevent the diacylglycerol-induced reduction of junctional permeability. Nor did sodium orthovanadate (25 or 50 m)—an inhibitor of tyrosyl phosphatase-prevent the reversal of diacylglycerol-induced (or TPA-induced) reduction of junctional permeability.     

Homologous and heterologous cell coupling in mammalian ovarian follicles

Movement of markers derived from ³H-labelled choline through the intact membrana granulosa has been quantified using scintillation counting. This passage is most readily explained in terms of junctional transfer and may be detected across in excess of 400 cell diameters. Whereas gonadotropins reduce the movement of choline between cumulus cells and the oocyte, that between somatic cells is unaffected. Both CO₂ and the calcium-transporting ionophore A23187 markedly reduce movement of labelled components and the reported effects of CO₂ and Ca²⁺ support the conclusions that the gradients are a result of junctional transfer. In addition, morphological examination of CO₂-treated follicles shows a loss of gap junctions.     

Relations between d-ribulose-1,5-bisphosphate carboxylase,carboxysomes and CO2 fixing capacity in the obligate chemolithotroph Thiobacillus neapolitanus grown under different limitations in the chemostat

An adaptation of the d-ribulose-1,5-bisphosphate carboxylase (RuBPCase) activity to changing CO₂ concentrations in the growth medium in the chemostat was observed in the obligate chemolithotroph Thiobacillus neapolitanus. RuBPCase activity has been separated in a soluble and particulate fraction. The activity of the particulate fraction appeared to be associated with the carboxysomes.The total activity of RuBPCase of CO₂ limited cultures was about 5-fold higher than the activity of thiosulphate limited cultures grown in the presence of 5% CO₂ whilst the particulate activity and the soluble activity were about 8- and 1.5-fold higher, respectively. The fluctuation of the total and particulate RuBPCase activity correlated with the changes in volume density of carboxysomes in the cell.An inverse correlation between maximal CO₂ fixing capacity by whole cells and the volume density of carboxysomes was observed. The change in ratio of soluble RuBPCase activity to particulate RuBPCase activity paralleled the change in maximal CO₂ fixation by whole cells during the different growth conditions.     

Primary and long term epithelial cell cultures from human fetal normal colonic mucosa

Summary Primary and passaged cultures of normal colon epithelial cells, derived from human fetuses (13 to 17 wk of conceptual age) have been established. These cultures have been passaged 16 times thus far. The cultures have been initiated and maintained in medium consisting of 50% Dulbecco's minimum essential medium and 50% Ham's F12 medium and supplemented with antibiotics (penicillin, 100 U/ml; streptomycin, 100 μg/ml); ascorbic acid, 40 μg/ml;l-isoleucine, 50 μg/ml; epidermal growth factor, 20 ng/ml; insulin, 5 μg/ml; cholera toxin, 5 ng/ml; transferrin, 1 μg/ml; fetal bovine serum (10%); and HEPES, 25 mM final concentration, and incubated at 37°C in humidified gas containing 5% CO₂: 95% air. The cellular and subcellular characteristics of primary and passaged cultures were defined using light microscopy and scanning and transmission electron microscopy. The cells exhibited microvilli on cell surfaces and showed junctional complexes and interdigitations between cells. Indented nuclei with dense chromatin and marginated heterochromatin, numerous mitochondria, rough endoplasmic reticulum, polysomes, and extensive Golgi zones were conspicuous. Also, periodic acid Schiff's reagent-positive staining of the cells suggests the active synthesis of complex mucopolysaccharides in the cytoplasm. This study was supported by USPHS Grant CA-30185 from the National Large Bowel Cancer Project, National Cancer Institute.     

Caffeine inhibition of calcium accumulation by the sarcoplasmic reticulum in mammalian skinned fibers

Summary Oxalate-supported Ca accumulation by the sarcoplasmic reticulum (SR) of chemically skinned mammalian skeletal muscle fibers is activated by MgATP and Ca²⁺ and partially inhibited by caffeine. Inhibition by caffeine is greatest when Ca²⁺ exceeds 0.3 to 0.4 m, when free ATP exceeds 0.8 to 1mm, and when the inhibitor is present from the beginning of the loading period rather than when it is added after Ca oxalate has already begun to precipitate within the SR. Under the most favorable combination of these conditions, this effect of caffeine is maximal at 2.5 to 5mm and is half-maximal at approximately 0.5mm. For a given concentration of caffeine, inhibition decreases to one-half of its maximum value when free ATP is reduced to 0.2 to 0.3mm. Varying free Mg²⁺ (0.1 to 2mm) or MgATP (0.03 to 10mm) has no effect on inhibition. Average residual uptake rates in the presence of 5mm caffeine atpCa 6.4 range from 32 to 70% of the control rates in fibers from different animals. The extent of inhibition in whole-muscle homogenates is similar to that observed in skinned fibers, but further purification of SR membranes by differential centrifugation reduces their ability to respond to caffeine. In skinned fibers, caffeine does not alter the Ca²⁺ concentration dependence of Ca uptake (K _0.5, 0.5 to 0.8 m; Hilln, 1.5 to 2.1). Reductions in rate due to caffeine are accompanied by proportional reductions in maximum capacity of the fibers, and this configuration can be mimicked by treating fibers with the ionophore A23187. Caffeine induces a sustained release of Ca from fibers loaded with Ca oxalate. However, caffeine-induced Ca release is transient when fibers are loaded without oxalate. The effects of caffeine on rate and capacity of Ca uptake as well as the sustained and transient effects on uptake and release observed under different conditions can be accounted for by a single mode of action of caffeine: it increases Ca permeability in a limited population of SR membranes, and these membranes coexist with a population of caffeine-insensitive membranes within the same fiber.     

Modulation of apical Na permeability of the toad urinary bladder by intracellular Na,Ca, and H

Summary The Na conductance of the apical membrane of the toad urinary bladder was measured at different concentrations of Na both in the external medium and in the cell. Bladders were bathed in high K-sucrose medium to reduce basal-lateral resistance and voltage, and the transepithelial currents measured under voltage-clamp conditions. Amiloride was used as a specific blocker of the apical Na channel. At constant external Na, the internal Na concentration was increased by blocking the basallateral Na pump with ouabain. With high Na activity in the mucosal medium (86mm), increases in intracellular Na activity from 10 to over 40mm increased the amiloride-sensitive slope conductance at zero voltage while apical Na permeability, estimated from current-voltage plots using the constant field equation, decreased by less than 20%. Lowering the serosal Ca concentration from 1 to 0.1mm had no effect on the change inP _Na with increasing Na_c, but increasing serosal Ca to 5mm enhanced the reduction inP _Na with increasing Na _c , presumably by increasing Ca influx into the cell.P _Na was also reduced by serosal vanadate (0.5mm), a putative blocker of ATP-dependent Ca extrusion from the cell, and by acute exposure to CO₂, which presumably acidifies the cytoplasm. Current-voltage relationships of the amiloridesensitive transport pathway were also measured in the absence of a Na gradient across the apical membrane. These plots show that outward current passes through the channels somewhat less easily than does inward current. The shape of theI-V relationships was not significantly altered by changes in cellular Na, Ca or H, indicating that the effects of these ions onP _Na are voltage independent.     

Intracellular pH,intracellular free Ca,and junctional cell-cell coupling

Summary Intracellular pH (pH _i ) and intracellular Ca²⁺ ([Ca²⁺] _i ) were determined inChironomus salivary gland cells under various conditions of induced uncoupling. pH _i was measured with aThomas-type microelectrode, changes in [Ca²⁺] _i and their spatial distribution inside the cell were determined with the aid of intracellularly injected aequorin and an image intensifier-TV system, and cell-to-cell coupling was measured electrically. Treatments with NaCN (5mm), DNP (1.2mm), or ionophore A23187 (2m) caused fall in junctional conductance (uncoupling) that was correlated with [Ca²⁺] _i elevation, as was shown before (Rose & Loewenstein, 1976,J. Membrane Biol. 28:87) but not with changes in pH _i : during the uncoupling induced by CN, the pH _i (normally 7.5) decreased at most by 0.2 units; during the uncoupling induced by the ionophore, pH _i fell by 0.13 or rose by 0.3; and in any one of these three agents' uncouplings, the onset of uncoupling and recovery of coupling were out of phase with the changes in pH _i . Intracellular injection of Ca-citrate or Ca-EGTA solutions buffered to pH 7.2 or 7.5 produced uncoupling with little or no pH _i change when their free [Ca²⁺] _i was >10^–5 m. On the other hand, such a solution at pH 4, buffered to [Ca²⁺]<10^–6 m, lowered pH _i to 6.8 but produced no uncoupling. Thus, a decrease in pH _i is not necessary for uncoupling in any of these conditions. In fact, uncoupling ensued also during increase in pH _i : exposure to NH₄HCO₃ or withdrawal of propionate following exposure to a propionate-containing medium caused pH _i to rise to 8.74, accompanied by [Ca²⁺] _i elevation and uncoupling at pH _i >7.8.Cell acidification itself can cause elevation of [Ca²⁺] _i : injection (iontophoresis) of H⁺ invariably caused [Ca²⁺] _i elevation and uncoupling. These effects were produced also by an application of H⁺-transporting ionophore Nigericin at extracellular pH 6.5 which caused pH _i to fall to 6.8. Exposure to 100% CO₂ produced a fall in pH _i , associated in 10 out of 25 cases with [Ca²⁺] _i elevation and, invariably, with uncoupling. The absence of a demonstrable [Ca²⁺] _i elevation in a proportion of these trials is attributable to depression in Ca²⁺-measuring sensitivity; inin vivo tests, detection sensitivity for [Ca²⁺] _i by aequorin was found to be depressed by the CO₂ treatment. Upon CO₂ washout, pH _i and coupling recovered, but onset of recoupling set in at pH _i as low as 6.32–6.88, generally lower than at the pH _i at which uncoupling had set in. Exposure to 5% CO₂ lowered pH _i on the average by 0.3 and depressed coupling (in initially poorly coupled cells). After CO₂-washout, pH _i and coupling recovered. During the recovery phase [Ca²⁺] _i was elevated, an elevation associated with renewed uncoupling or decrease in rate of recoupling. The results are discussed in connection with possible regulatory mechanisms of junctional permeability.     

Cyclic AMP induces rapid increases in gap junction permeability and changes in the cellular distribution of connexin43

The rapid effects of cAMP on gap junction-mediated intercellular communication were examined in several cell types which express different levels of the gap junction protein, connexin43 (Cx43), including immortalized rat hepatocyte and granulosa cells, bovine coronary venular endothelial cells, primary rat myometrial and equine uterine epithelial cells. Functional analysis of changes in junctional communication induced by 8-bromo-cAMP was monitored by a fluorescence recovery after photobleaching assay in subconfluent cultures in the presence or absence of 1.0 mm 1-octanol (an agent which uncouples cells by closing gap junction channels). Communicating cells treated with 1.0 mm 8-bromo-cAMP alone exhibited significant increases in the percent of fluorescence recovery which were detected within 1–3 min depending on cell type, and junctional communication remained significantly elevated for up to 24 hr. Addition of 1.0 mm 8-bromo-cAMP to cultured cells, which were uncoupled with 1.0 mm octanol for 1 min, exhibited partial restoration of gap junctional permeability beginning within 3–5 min. Identical treatments were performed on cultures that were subsequently processed for indirect immunofluorescence to monitor Cx43 distribution. The changes in junctional permeability of cells correlated with changes in the distribution of immunoreactive Cx43. Cells treated for 2 hr with 10 m monensin exhibited a reduced communication rate which was accompanied by increased vesicular cytoplasmic Cx43 staining and reduced punctate surface staining of junctional plaques. Addition of 1.0 mm 8-bromo-cAMP to these cultures had no effect on the rate of communication or the distribution of Cx43 compared to cultures treated with monensin alone. These data suggest that an effect of cyclic AMP on Cx43 gap junctions is to promote increases in gap junctional permeability by increasing trafficking and/or assembly of Cx43 to plasma membrane gap junctional plaques.We acknowledge the technical assistance of Richard Lewis and Meghan Abella. We thank Dr. Hugh Dookwah for contributions to the myometrial cell isolation protocol and Drs. Stephen H. Safe, Timothy D. Phillips, and Evelyn Tiffany-Castiglioni for helpful discussions. This work was funded by NIH (HD-26182, P42-ES04917, ES05871-01A1), the March of Dimes Birth Defects Foundation Basic Research grant #1-0796, and USDA 92-37203-7952.     

Charybdotoxin blocks with high affinity the Ca-activated K+ channel of Hb A and Hb S red cells: Individual differences in the number of channels

Summary We have investigated the effect of a purified preparation of Charybdotoxin (CTX) on the Ca-activated K⁺ (Ca–K) channel of human red cells (RBC). Cytosolic Ca²⁺ was increased either by ATP depletion or by the Ca ionophore A23187 and incubation in Na⁺ media containing CaCl₂. The Ca–K efflux activated by metabolic depletion was partially (77%) inhibited from 15.8±2.4 mmol/liter cell · hr, to 3.7±1.0 mmol/liter cell · hr by 6nm CTX (n=3). The kinetic of Ca–K efflux was studied by increasing cell ionized Ca²⁺ using A23187 (60 mol/liter cell), and buffering with EGTA or citrate; initial rates of net K⁺ efflux (90 mmol/liter cell K⁺) into Na⁺ medium containing glucose, ouabain, bumetanide at pH 7.4 were measured. Ca–K efflux increased in a sigmoidal fashion (n of Hill 1.8) when Ca²⁺ was raised, with aK _m of 0.37 m and saturating between 2 and 10 m Ca²⁺. Ca–K efflux was partially blocked (71±7.8%, mean ±sd,n=17) by CTX with high affinity (IC₅₀0.8nm), a finding suggesting that is a high affinity ligand of Ca–K channels. CTX also blocked 72% of the Ca-activated K⁺ efflux into 75mm K⁺ medium, which counteracted membrane hyperpolarization, cell acidification and cell shrinkage produced by opening of the K⁺ channel in Na⁺ media. CTX did not block Valinomycin-activated K⁺ efflux into Na⁺ or K⁺ medium and therefore it does not inhibit K⁺ movement coupled to anion conductive permeability.TheV _max, but not theK _m–Ca of Ca–K efflux showed large individual differences varying between 4.8 and 15.8 mmol/liter cell · min (FU). In red cells with Hb A,V _max was 9.36±3.0 FU (mean ±sd,n=17). TheV _max of the CTX-sensitive, Ca–K efflux was 6.27±2.5 FU (range 3.4 to 16.4 FU) in Hb A red cells and it was not significantly different in Hb S (6.75±3.2 FU,n=8). Since there is larger fraction of reticulocytes in Hb S red cells, this finding indicates that cell age might not be an important determinant of theV _max of Ca–K⁺ efflux.Estimation of the number of CTX-sensitive Ca-activated K⁺ channels per cell indicate that there are 1 to 3 channels/per cell either in Hb A or Hb S red cells. The CTX-insensitive K⁺ efflux (2.7±0.9 FU) may reflect the activity of a different channel, nonspecific changes in permeability or coupling to an anion conductive pathway.     

Muscarinic responses of gastric parietal cells

Summary Isolated rabbit gastric glands were used to study the nature of the muscarinic cholinergic responses of parietal cells. Carbachol (CCh, 100 m) stimulation of acid secretion, as measured by the accumulation of aminopyrine, was inhibited by the M1 antagonist, pirenzepine, with an IC₅₀ of 13 m; by the M2 antagonist, 11,2-(diethylamino)methyl-1 piperidinyl acetyl-5,11-dihydro-6H-pyrido 2,3-b 1,4 benzodiazepin-6-one (AF-DX 116), with an IC₅₀ of 110 m; and by the M1/M3 antagonist, diphenylacetoxy-4-methylpiperidinemethiodide (4-DAMP), with an IC₅₀ of 35nm. The three antagonists displayed equivalent IC₅₀ values for the inhibition of carbachol-stimulated production of¹⁴CO₂ from radiolabeled glucose, which is a measure of the turnover of the H,K-ATPase, the final step of acid secretion. Intracellular calcium levels were measured in gastric glands loaded with FURA 2. Carbachol was shown to both release calcium from an intracellular pool and to promote calcium entry across the plasma membrane. The calcium entry was inhibitable by 20 m La³⁺. The relative potency of the three muscarinic antagonists for inhibition of calcium entry was essentially the same as for inhibition of acid secretion or pump related glucose oxidation. Image analysis of the glands showed the effects of carbachol, and of the antagonists, on intracellular calcium were occurring largely in the parietal cell. The rise in cell calcium due to release of calcium from intracellular stores was inhibited by 4-DAMP with an IC₅₀ of 1,7nm, suggesting that the release pathway was regulated by a low affinity M3 muscarinic receptor or state; Ca entry and acid secretion are regulated by a high affinity M3 muscarinic receptor or state, inhibited by higher 4-DAMP concentrations (>30nm), suggesting that it is the steady-state elevation of Ca that is related to parietal cell function rather than the [Ca] _i transient. Displacement of³H N-methyl scopolamine (NMS) binding to purified parietal cells by CCh showed the presence of two affinities for CCh, but only a single affinity for 4-DAMP and lower affinity for pirenzepine and AFDX 116, providing further evidence for the parietal cell location of the [Ca] _i response. Elevation of steady-state [Ca] _i levels with either ionomycin or arachidonic acid did not replicate M3 stimulation of acid secretion or glucose oxidation, hence elevation of [Ca] _i is necessary but not sufficient for acid secretion.     

Clostridium methylpentosum sp. nov.: a ring-shaped intestinal bacterium that ferments only methylpentoses and pentoses

An intestinal bacterium isolated from a human subject utilized only two methylpentoses (L-rhamnose and L-fucose) and two pentoses (L-lyxose and D-arabinose) as fermentable substrates, among many compounds tested. The isolate was obligately anaerobic and had a distinctive morphology, its cells being rods bent in the shape of rings with the ends slightly overlapping. Single ring-shaped cells and left-handed helical chains of cells were present in cultures. The cells were surrounded by large capsules which appeared as thick, fibrous masses when examined by electron microscopy. Capsules were formed by cells growing in media containing any one of the four fermentable substrates. Terminally located, heat-resistant endospores were formed on plates of an enriched agar medium supplemented with L-rhamnose. End products of L-rhamnose or L-fucose fermentation included acetate, propionate, n-propanol, CO₂, and H₂. The isolate represented a new species of Clostridium for which the name Clostridium methylpentosum (type strain R2. ATCC 43829) is proposed. This organism may participate in intestinal digestive processes by metabolizing rhamnose released via the enzymatic depolymerization of dietary pectin.Abbreviations G+C guanine plus cytosine - OD optical density - TEM transmission electron micrograph     

Ethanol production during D-xylose,L-arabinose,and D-ribose fermentation by Bacteroides polypragmatus

Summary The specific growth rate () during cultivation of Bacteroides polypragmatus in 2.51 batch cultures in 4–5% (w/v) l-arabinose medium was 0.23 h^-1 while that in either d-xylose or d-ribose medium was lower (=0.19 h^-1). Whereas growth on arabinose or xylose occurred after about 6–8 h lag period, growth on ribose commenced after a 30 h lag phase. The maximum substrate utilization rate for arabinose, ribose and xylose in media with an initial substrate concentration of 4–5% (w/v) was 0.77, 0.76, and 0.60 g/l/h respectively. In medium containing a mixture of glucose, arabinose, and xylose, the utilization of all three substrates occurred concurrently. The maximum amount of ethanol produced after 72 h growth in 4–5% (w/v) of arabinose, xylose, and ribose was 9.4, 6.5, and 5.3 g/l, respectively. The matabolic end products (mol/mol substrate) of growth in 4.4% (w/v) xylose medium were 0.73 ethanol, 0.49 acetate, 1.39 CO₂, 1.05 H₂, and 0.09 butyrate.National Research Council of Canada No. 23406     

Photoautotrophic Growth and Photosynthesis in Cell Suspension Cultures of Chenopodium rubrum

A method is described for growing cell suspension cultures of Chenopodium rubrum photoautotrophically for prolonged periods of time. By using a two-tier culture vessel the growth medium with the cells was separated from the CO₂ reservoir. Definite CO₂ concentrations were established by a K₂CO₃/KHCO₃ buffer. Photoautotrophic growth in C. rubrum cell suspension cultures was correlated with the CO₂ level. At 0.5% CO₂ the cell cultures contained 68 μg chlorophyll/g fresh weight and showed an increase in fresh weight of about 80% in 18 days. At 1% CO₂ an increase in fresh weight of 165% in 18 days was observed. The chlorophyll content rose up to 84 μg/g fresh weight. The photoautotrophic growth was also greatly influenced by the 2,4-D content of the medium. Cell growth was enhanced by lowering the auxin concentration. Best growth was attained (210% increase in fresh weight) at 10^?8M 2,4-D. The photosynthetic activity of the cells was measured by the light dependent ¹⁴CO₂ incorporation. At 0.5% CO₂ the cell suspensions assimilated about 100 μmol CO₂/mg chlorophyll × h. In the presence of 1% CO₂ the light driven assimilation was raised up to 185 μmol CO₂/mg chlorophyll × h. In both cases, the dark incorporation of CO₂ was merely 1.8% of the values obtained in light.     

Comparative metabolic effects of fructose and glucose in human fibroblast cultures

Summary The comparative metabolic effects of fructose and glucose were determined in human fibroblast cultures. Cells were grown in four different media containing 5.5 and 27.5 mM of glucose and fructose, respectively. For these two hexoses, we compared their uptake, consumption, and conversion into¹⁴CO₂ and¹⁴C-lipids. D-Fructose was taken up in fibroblasts by an unsaturable process and its consumption was much smaller than that ofD-glucose. Whatever the experimental procedure, the glycogen content of cells grown in fructose media was significantly lower than of those grown in glucose media. Labeling of fructose and glucose with¹⁴C showed that more carbon from fructose than from glucose was incorporated into CO₂ and glycerolipids. The relative distribution of¹⁴C in the different lipid fractions was similar for both hexoses. These results indicated that the pathways of intermediary metabolism in fibroblast cultures were influenced by the nature of the carbohydrate present in the culture medium and that fructose was a better lipogenic substrate than glucose in human fibroblast cultures. This work was supported by grants for the Institut National de la Santé et de la Recherche Medicale (ATP 82-79-114).     

Bicarbonate and fast-twitch muscle: Evidence for a major role in pH regulation

Summary Internal pH (pH_i) was analyzed in rat extensor digitorum longus (Edl) muscle at 30°C with single-barrel liquid ionselective electrodes. Average pH_i in 284 cells was 7.197±0.006. Increases in CO₂ from nominally 0 to 5% produced an acidification from which recovery took place. In different groups of cells, recovery from the 5% CO₂ acidification was significantly inhibited by 100 m 4,4 diisothiocyanatostilbene 2,2 disulfonic acid (DIDS), Cl removal, Na removal and 2mm amiloride. Prepulsing with 20mm NH₄ in the presence of CO₂/HCO₃ typically reduced pH_i to only about neutral, whereas 50mm reduced pH_i to 6.7–6.8. In the nominal absence of CO₂/HCO₃, 20mm NH₄ reduced pH_i to about 6.7 from which recovery took place at about 58% of the rate seen in different cells in the presence of CO₂/HCO₃. In the presence of CO₂/HCO₃, cells prepulsed with 50mm NH₄ had fully recovered to an average pH_i of 7.22±0.04 about 90 min after removal of NH₄. However, 90 min after removal of 20mm NH₄ in the absence of CO₂/HCO₃, average pH_i was significantly less (7.05±0.03). Intrinsic buffering capacity ( _i ) was obtained during pulses of CO₂, acetic acid or after an NH₄ pulse, _i was significantly reduced in the absence of HCO₃, Cl or Na and HCO₃. The data provide significant support for an important role of HCO₃ in the control of pH_i in fast-twitch muscle.     

Electrical coupling between cells in islets of langerhans from mouse

Summary Two microelectrodes have been used to measure membrane potentials simultaneously in pairs of mouse pancreatic islet cells. In the presence of glucose at concentrations between 5.6 and 22.2mm, injection of currenti into cell 1 caused a membrane potential change in this cell,V ₁, and, provided the second microelectrode was less than 35 m away, in a second impaled cell 2,V ₂. This result establishes that there is electrical coupling between islet cells and suggests that the space constant of the coupling ratio within the islet tissue is of the order of a few -cell diameters. The current-membrane potential curvesi-V ₁ andi-V ₂ are very similar. By exchange of the roles of the microelectrodes, no evidence of rectification of the current through the intercellular pathways was found. Removal of glucose caused a rapid decrease in the coupling ratioV ₂ /V ₁ . In steady-state conditions, the coupling ratio increases with the concentration of glucose in the range from 0 up to 22mm. Values of the equivalent resistance of the junctional and nonjunctional membranes have been estimated and found to change with the concentration of glucose. Externally applied mitochondrial blockers induced a moderate increase in the junctional resistance possibly mediated by an increase in intracellular Ca²⁺.     

The chloride concentration in the lateral intercellular spaces of MDCK cell monolayers

We measured the Cl concentration of the lateral intercellular spaces (LIS) of MDCK cell monolayers, grown on glass coverslips, by video fluorescence microscopy. Monolayers were perfused at 37°C either with HEPES-buffered solutions containing 137 mm Cl or bicarbonate/CO₂-buffered solutions containing 127 mm Cl. A mixture of two fluorescent dyes conjugated to dextrans (MW 10,000) was microinjected into domes and allowed to diffuse into the nearby LIS. The Cl sensitive dye, ABQ-dextran, was selected because of its responsiveness at high Cl concentrations; a Clinsensitive dye, Cl-NERF-dextran, was used as a reference. Both dyes were excited at 325 nm, and ratios of the fluorescence intensity at spectrally distinct emission wavelengths were obtained from two intensified CCD cameras, one for ABQ-dextran the other for Cl-NERFdextran. LIS Cl concentration was calibrated in situ by treating the monolayer with digitonin or ouabain and varying the perfusate Cl between 0 and 137 mm (HEPES buffer) or between 0 and 127 mm (bicarbonate/CO₂ buffer). LIS Cl in HEPES-buffered solutions averaged 176 ± 19 mm (n = 12), calibrated with digitonin, and 170 ± 9 mm (n = 12), calibrated with ouabain. LIS Cl in bicarbonate/CO₂-buffered solutions averaged 174 ± 10 mm (n = 7) using the ouabain calibration. The Cl concentration of MDCK cell domes, measured with Clsensitive microelectrodes and by microspectrofluorimetry, did not differ significantly. Images of the LIS at 3 focal planes, near the tight junction, midway and basal, failed to reveal any gradients in Cl concentration along the LIS. LIS Cl changed rapidly in response to perfusate Cl with characteristic times of 0.8 ± 0.1 min (n = 21) for Cl decrease and 0.3 ± 0.04 min (n=21) for Cl increase. In conclusion, (i) Cl concentration is higher in the LIS than in the bathing medium, (ii) no gradients of Cl along the depth of LIS are detectable, (iii) junctional Cl permeability is high.We gratefully acknowledge the assistance of Mr. Richard D'Alessandro in the performance of the microelectrode studies. Mr. Carter Gibson designed the electronics and wrote the key computer programs used in this study. The authors are grateful to Dr. Alan Verkman (UCSF) for his advice and gifts of fluorescent probes in the early stages of this work.