Contraluminal transport of hexoses in the proximal convolution of the rat kidney in situ

In order to study contraluminal hexose transport, concentration and time-dependent influx of³H-2-deoxy-d-glucose from the interstitium into cortical tubular cells has been measured. The influx curves fit to a two parameter kinetics (K _m 1.3±0.2 mmol/l,J _max 0.67±0.16 pmol/s · cm) plus an additional diffusion term (withP=6·10^–8 cm²/s) and a distribution ratio extracellular to intracellular amount of 2-deoxy-d-glucose of 10.6. Since the extracellular to intracellular free water space as estimated from morphological data was 12, one must conclude that glucose has only free access to 1/3 of the cell water. The intracellularly accessible space was augmented when the tubules were preperfused for 10 s with hypotonic saline. Thereby an increase of the compartment into which diffusion occurs was revealed and a final rupture of this intracellular compartment at 1/4 isotonic solutions was observed. Total replacement of ions in the peritubular perfusate by mannitol did not change 2-deoxy-d-glucose influx, indicating that it is Na⁺-independent. By adding isotonic concentrations of the respective sugars to the capillary perfusate, three degrees of inhibition of 2-deoxy-d-glucose influx could be revealed: strong inhibition byd-glucose, methyl--d-glucoside,d-mannose, 3-O-methyl-d-glucose, 2-deoxy-d-galactose, methyl--d-galactoside and 6-deoxy-d-glucose, moderate inhibition byd-galactose,l-glucose,l-mannose andd-fructose, no or borderline inhibition by methyl -d-glucoside, 2-deoxy-methyl--d-galactoside, 1-thio--d-glucose, 1-thio--d-galactose, 5-thio--d-glucose, myo-inositol and mannitol. The contraluminal 2-deoxy-d-glucose influx was also inhibited by phloretin, chlormerodrin and preperfusion with cytochalasin B. Starvation as well as streptozotocin diabetes has no influence on contraluminal 2-deoxy-d-glucose transport. Thus, in contrast to the luminal hexose transport system the contraluminal system is Na⁺-independent, does not require on OH-group at C-atom 2, acceptsl-glucose and fructose, but not an -methyl group at C-atom 1.     

Studien über den Mechanismus der Immunhämolyse: Der Bindungsmodus der vierten Komplementkomponente

Zusammenfassung Die Bindung der vierten Komplementkomponente (C₄) an sensibilisierte Hammelblutzellen (EA) ist von der vorhergehenden Bindung der ersten Komplementkomponente abhängig. Das Präparat EAC₁ kann, wenn ihm C₄ in geeigneter Form angeboten wird, in den Komplex EAC_1,4 überführt werden. Hierbei verschwindet der titrierbare Gehalt der flüssigen Phase an C₄; gleichzeitig acquirieren die Zellen die Fähigkeit, mit R₄ zu lysieren. Der zeitliche Ablauf dieser Veränderung wird untersucht. Die Eeaktion zwischen EAC₁ und C₄ verläuft äußerst schnell, und ihre Geschwindigkeit wird durch die Reaktionstemperatur nicht beeinflußt. Während die Bindung von C₁ an EA nur in Gegenwart von Ca⁺⁺ erfolgt, läuft die Bindung von C₄ an EAC₄ auch in Abwesenheit von zweiwertigen Metallionen ab. Die vonLevine u.Mayer als ein Ganzes betrachtete Ca⁺⁺-abhängige Überführung von EA in EAC_1,4 besteht mithin aus zwei aufeinanderfolgenden Teilreaktionen, von denen die erste (C₁-Bindung) calciumabhängig und die zweite (C₄-Bindung) von zweiwertigen Ionen unabhängig ist.Mit Unterstützung der Deutschen Forschungsgemeinschaft sowie der Gesellschaft der Freunde und Förderer der Medizinischen Akademie Düsseldorf.     

Evidence for Na/H exchange and Cl/HCO3 exchange in A10 vascular smooth muscle cells

In the present study we used the pH sensitive absorbance of 5(and6)-carboxy-4,5-dimethylfluorescein to investigate intracellular pH (pH_i) regulation in A10 vascular smooth muscle cells: (1) The steady state pH_i in A10 cells averaged 7.01±0.1 (mean±SEM,n=26) at an extracellular pH of 7.4 (28 mM HCO₃/5% CO₂). (2) Removal of extracellular sodium led to an intracellular acidification of 0.36±0.07 pH-units (mean±SEM,n=8). (3) pH_i-Recovery after an acute intracellular acid load (by means of NH₄Cl-prepulse) was reversibly blocked by 1 mM amiloride and was dependent on the presence of sodium. The velocity of pH_i recovery increased with increasing sodium concentrations with an apparentK _m for external sodium of about 30 mM and aV _max of about 0.35 pH units/min. These findings are compatible with a Na/H exchanger being responsible for pH_i recovery after an acid load. (4) Removal of extracellular chioride induced an intracellular alkalinization of 0.23±0.03 pH-units (mean±SEM,n=10). The alkalinization was dependent on the presence of extracellular bicarbonate (5) Removal of chloride during pH_i recovery from an alkaline load (imposed by acetate prepulse) stopped and reversed pH_i backregulation. Chloride removal had no effect in the absence of bicarbonate or in the presence of 10^–4 M DIDS, suggesting that the effects were mediated by a Cl/HCO₃ exchanger. In conclusion we have demonstrated evidence for a Na/H exchanger and a Cl/HCO₃ exchanger in A10 vascular smooth muscle cells.Abbreviations used CDMF 5(and6)-carboxy-4,5-dimethylfluorescein - DIDS 4,4-diisothiocyanostilbene-2,2-disulfonic acid - NMDG N-methyl-d-glucamine; pH_i, intracellular pH - pH_o extracellular pH - Mops 3-[N-Morpholino]propanesulfonic acid - Hepes 2-[4-(2-Hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid - Tris Tris(hydroxymethyl)-aminomethane - EDTA ethylenediamine-tetraacetic acid - EGTA ethyleneglycol-bis-(-amino-ethylether)N,N-tetraacetic acid     

45Ca2+ uptake by dispersed pancreatic islet cells: Effects ofd-glucose and the calcium probe,chlorotetracycline

Uptake of⁴⁵Ca²⁺ was studied in dispersed pancreatic islet cells from non-inbredob/ob-mice. Like whole islets the dispersed cells responded to 20 mMd-glucose with a markedly increased⁴⁵Ca²⁺-labeling of both the lanthanum-nondisplaceable and the lanthanum-displaceable calcium pools. The pronounced effect ofd-glucose could not be reproduced with 3-O-methyl-d-glucose,l-glucose,d-mannose,l-leucine, ord-leucine; however,⁴⁵Ca²⁺ uptake was greater in the presence ofl-leucine as compared withd-leucine.⁴⁵Ca²⁺ uptake by dispersed cells or whole islets was stimulated severalfold by 100 M or more chlorotetracycline. At the concentration of only 10 M, chlorotetracycline had no effect on whole islets and partially inhibited⁴⁵Ca²⁺ uptake by the dispersed cells. The ability ofd-glucose to stimulate⁴⁵Ca²⁺ uptake by islets or dispersed cells remained in the presence of 10 M chlorotetracyline. Islet cell suspensions apparently represent a valid model for studying how Ca²⁺ interacts with the cells. However, when using chlorotetracycline as fluorescent Ca²⁺ probe, attention must be paid to its potential ionophoric activity. At only 10 M, the drug seems to monitor a peripheral pool of Ca²⁺, some of which may reside in normal transport channels.     

d-glucose balance in the enterocyte of rat jejunum “in vitro”

An everted sac of male albino rat jejunum (Wistar strain) incubated in vitro is used. Netd-glucose and Na⁺ transport together withd-glucose concentration in the emerging fluid [5], or in the serosal fluid, and in the enterocytes are determined. Celld-glucose concentration does not change significantly in a range between 20–200 moles or between 50–500 moles of netd-glucose transepithelial transport, depending on the experimental conditions.As far as cellulard-glucose and Na⁺ concentration is concerned, the enterocyte behaves as an homeostatic system.The mechanism involved ind-glucose extrusion is extensively discussed. Two hypotheses seem to be possible. First, the mechanism is an active metabolically dependent one, just as it is for sodium transport. Second, the metabolic activity favoursd-glucose facilitated permeability through the basolateral membrane in such a way as to maintain a constant relationship betweend-glucose and Na-extrusion, notwithstanding the fact thatd-glucose concentration gradient across the basolateral membrane lowers by increasing Na and glucose extrusion rate.     

Molecular specificity of the tubular resorption of “acidic” amino acids

Single sections of superficial proximal convolutions of rat kidney were microperfused in vivo and in situ. The perfusion fluids contained radioactively labelledl- ord-aspartate,l-glutamate,l-pyroglutamate, or N-methyl-d-aspartate.l--Carboxyglutamate as well as the other amino acids were added in the unlabelled from. Results.l- andd-Aspartate (0.073 mmol·1^–1) are quickly resorbed at about the same rate.d-Aspartate resorption was blocked byl-aspartate (5 mmol·1^–1) but not by -alanine (5 mmol·1^–1).l-Aspartate resorption was inhibited byl-glutamate (2 mmol·1^–1) but not byd-glutamate,l-asparagine,l-phenylalanine or by succinate (2 mmol·1^–1, each). The fast resorption ofl-glutamate (0.073 mmol·1^–1) was blocked byd-aspartate,l-cysteate (2 mmol·1^–1), but not by 3-mercaptopicolinic acid (0.15 mmol·1^–1),l-glutamine, 2-oxoglutarate, taurine, N-methyl-l-glutamate or kainic acid (2 mmol·1^–1, each).l--Carboxyglutamate (0.66 mmol·1^–1) and N-methyl-d-aspartate (2mol·1^–1) were found to be resorbed only at an extremely small rate.l-pyroglutamate (0.076 mmol·1^–1) resorption was not influenced byl-glutamate (1 mmol·1^–1). Fractional excretion of -carboxyglutamate was 7–25% (l-from) or 45–70% (d-form) at an artificially elevated plasma level of 12mol·1^–1.It is concluded thatl- andd-aspartate,l-glutamate,l-cysteate and, to a much smaller extent,l--carboxyglutamate, are accepted by the tubular resorption mechanism highly specific for acidic amino acids. N-Substitution, the amidation of the - or -carboxyl group, or the removal of the -amino moiety almost completely abolish the ability of such compounds to be resorbed via this carrier; N-methylated or -carboxylated derivatives of acidic amino acids are not resorbed at all from the proximal tubule. The resorption of glutamate, but not of aspartate, is highly stereospecific.Parts of this work were presented at meetings of the German Physiological Society in 1978 [28] and of the Gesellschaft für Nephrologie in 1980 [29] as well as at the VIIIth International Congress of Nephrology in Athens in 1981 [26]with technical assistance of Angelika Ascher and Gertaud Vetter     

Studien über den Mechanismus der Immunhämolyse: Die Bindung der zweiten Komplementkomponente

Zusammenfassung Es wird die Reaktion EAC_1,4 + C₂ isoliert untersucht. Hierzu bietet man einer Charge von stufenweise aufgebautem EAC_1,4 ein von allen übrigen Komplementkomponenten befreites C₂-Präparat an. Bei der Reaktion kommt es zu einem meßbaren Schwund von C₂ im Überstand; gleichzeitig erwerben die Zellen die Fähigkeit, mit cheliertem Komplement zu lysieren. Die Reaktionsgeschwindigkeit ist bei 37° C hoch und bei 0° C gering. Für die Fähigkeit der Zellen, C₂ zu binden, ist nicht allein das gebundene C₄ maßgebend, sondern ein von C₁ nicht abgrenzbarer Zusatzfaktor. Demnach kommen dem C₁ nach seiner Bindung zwei Funktionen zu: Einmal vermittelt es die Bindung von C₄, zum anderen vermittelt es zusammen mit dem gebundenen C₄ die Bindung von C₂.Mit Unterstützung der Deutschen Forschungsgemeinschaft sowie der Gesellschaft der Freunde und Förderer der Medizinischen Akademie Düsseldorf.     

Detection and quantitation of cell-surface sugar receptor(s) ofLeishmania donovani by application of neoglycoenzymes

Promastigote culture forms of the log growth phase ofLeishamania donovani stock LRC L 51 were investigated for expression of cell-surface carbohydrate-binding sites using 15 types of a chemically glycosylated enzyme termed neoglycoenzyme. Carbohydrate conjugation and coupling yield were kept constant to ensure that the type of carbohydrate moiety, was the only variable feature of the applied tools. Para-aminophenyl derivatives of the following carbohydrate residues were used for the glycosylation of -galactosidase fromEscherichia coli: -d-lactose, -d-thiogalactose, -d-mannose, -l-rhamnose, -d-N-acetylgalactosamine, -d-N-acetylgalactosamine, -d-N-acetylgalactosamine, -d-N-acetylgalactosamine, -d-N-acetylglucosamine, the - and -glucosides maltose and cellobiose, -d-xylose, -d-mannose-6-phosphate, the -galactoside melibiose, -l-fucose, and -d-glucuronic acid as well as sialic acid. Only melibiose, fucose, and glucuronic acid showed no binding affinity for the cultured flagellates; this served as an internal control reaction to exclude any binding to the linker group. This result demonstrates that many but not all sugar types can be recognized by appropriate receptor structure(s) on the surface of the promastigoteLeishmania. Transformation of the binding data for neoglycoenzymes exposing lactose, mannose, rhamnose, andN-acetylated hexose residues, which was carried out to obtain the dissociation constants and to estimate the number of binding sites at saturation, revealedK _D values of around 100mm and around 10⁴ binding sites for the polyvalent ligands.     

Endothelin-1 inhibitsl-type Ca currents enhanced by isoproterenol in guinea-pig ventricular myocytes

To investigate the action of Endothelin-1 (ET-1) on L-type Ca currents (I _Ca,L) in guinea-pig ventricular cells, whole-cell currents were recorded at 36-37° C in enzymatically isolated myocytes. ET-1 ( 10 nM) suppressed the basal I_ca,L to 79 ± 8% of control at 20 nM. Bath application of isoproterenol (ISO; 10 nM) enhanced I_Ca,L to 192±28% with about a – 10-mV shift of its relationship with membrane potential. ET-1 concentration dependently inhibited this ISO-enhanced I_Ca,L with a half-maximally inhibitory concentration (IC₅₀) of 168 pM. The inhibitory actions of ET-1 were antagonised by BQ-123 (300 nM), cyclo(d-Asp-l-Pro-d-Val-l-Leu-d-Trp), a specific ETA receptor antagonist. Histamine-enhancedI _Ca,L was also suppressed by ET-1, butI _Ca,L potentiated by internal adenosine 3,5-cyclic monophosphate (cAMP) was unaffected. Preincubation of myocytes with pertussis toxin (PTX, at 5 g/ml for >60 min at 36° C) completely occluded the ET-1 action. Thus, stimulation of ET_A receptors by subnanomolar ET-1 inhibitsI _Ca,L via PTX-sensitive G-proteins.     

Isolation and preliminary characterization of 9-β-d-arabinofuranosyladenine-resistant mutants of baby hamster cells

A large number of 9--d-arabinofuranosyladenine (araA) -resistant mutants of baby hamster kidney cells (BHK 21/Cl3) were isolated. These mutants can be grouped into three mechanistically distinct classes. All the mutants showed cross-resistance to deoxyadenosine (dAdo). The mechanism of resistance to araA and dAdo in the class I mutants can be attributed to a mutation to adenosine kinase (AK) deficiency. The class II mutants have normal levels of AK, adenosine deaminase, and deoxyadenosine kinase. These mutants also show resistance to 1--d-arabinofuranosylcytosine (araC), and the mechanism of resistance is probably due to a mutation in the ribonucleotide reductase gene producing an enzyme that has an increased resistance to the inhibition by 9--d-arabinofuranosyladenine 5-triphosphate (araATP) and 2-deoxyadenosine 5-triphosphate (dATP). The class III mutants, unlike those of classes I and II, show extreme adenosine (Ado) sensitivity. The Ado^s/araA^r/dAdo^r phenotypic properties can be attributed to a single mutation. Classes II and III are novel araA-resistant mutants.     

On the secretagogue effect of dibutyryl cyclic AMP in the rat exocrine pancreas

Summary DbcAMP0.1 mM induces the discharge of exportable enzymes from rat pancreas fragments incubated in vitro. This effect is qualitatively similar to the action of physiological secretagogues acting via hormone receptors: 1) it is accompanied by the appearance of exocytotic images at the acinar cell apex; 2) it is energy dependent but energy supply is low while that required for the carbamylcholine or caerulein response is high and can only be afforded by oxidative phosphorylation; 3) it is calcium dependent, but no alteration of inward or outward calcium movement can be observed; 4) it is altered by agents known to disrupt the microfilamentous microtubular system [41]. However, the secretory response to DbcAMP is quantitatively less than that obtained with hormonal stimuli. A damaging effect of DbcAMP on pancreatic acinar cells is ruled out on histological and biochemical grounds: there is no significant leakage of LDH; protein synthesis, 2-deoxy-d-glucose andl-leucine uptake are unaltered. The secretagogue effect of DbcAMP is reversible, dose-related and specific. It is not mediated by neuro-transmitter release or by interaction with their receptors. The evidence presented points to a direct interaction of DbcAMP on the pancreatic acinar cell and suggests the last step of the secretory cycle as the most probable site of action of the nucleotide derivative.Abbreviations cAMP Adenosine-3,5-monophosphate - cyclic DbcAMP: N⁶-2-O-Dibutyryl-adenosine-3,5-monophosphate, cyclic - DbcGMP N²-2-O-Dibutyryl-guanosine-3,5-monophosphate, cyclic - 5-AMP Adenosine-5-monophosphate - TCA Trichloracetic acid - ATP Adenosine triphosphate - NADH Nicotinamide-adenine dinucleotide - Tris Tris-(hydroxy-methyl) amino-methane - EGTA Ethylene glycol-bis ( aminoethylether)-NN-tetraacetic acid - LDH Lactic dehydrogenase Part of this work has been presented in abstract from at the VIIIth Symposium of the European Pancreatic Club, Toulouse, France, October, 1975, 23rd–25th and at the Biochemical Society of Belgium [40]This work was partially carried out under contracts from the Ministère de la Politique Scientifique within the framework of the Association Euratom—University of Brussels—University of Pisa, and the Institut National de la Santé et de la Recherche Médicale (France)     

Substrate utilization in the isolated perfused cortical thick ascending limb of rabbit nephron

Isolated segments of cortical thick ascending limbs (cTAL) of rabbit kidney were perfused in vitro and the equivalent short circuit current (Isc) was measured. In a first series all substrates were removed on either side. Isc fell rapidly to 50±12% after 3 min and to 27±6% (n=5) after 10 min. This indicates that in cTAL segments Isc is strictly dependent on the presence of substrates. In series two it was tested what substrates can be utilized by the cTAL segment, and from which epithelial side [bath (b) or lumen (l)] the substrates are taken up. From the l-side only butyrate (10 mmol · l^–1) sustained the Isc at 95±2% (n=7). All other tested substrates (10 mmol · l^–1): pyruvate, acetate, -OH-butyrate,d-glucose, andl-lactate lead to a marked decline in Isc. From the b-side several substrates (5–10 mmol · l^–1) sustained the Isc:d-glucose,d-mannose, butyrate, -OH-butyrate, acetoacetate,l-lactate, acetate and pyruvate. Other compounds (1–10 mmol · l^–1): citrate, -ketoglutarate, succinate, glutamate, glutamine, propionate, caprylate and oleate did not sustain Isc. In the third series the mechanism of substrate utilization from the basolateral cell side was studied. It was shown that the Isc is a saturable function of thed-glucose,l-lactate, acetate, pyruvate or -OH-butyrate concentration with apparentK _m's between 0.05–1.0 mmol · l^–1. Several known inhibitors of sugar and of anion transport were tested at the bath side: phlorrhizin was without effect. Phloretin (500 mol · l^–1) inhibited Isc by 96%, yet its effect was not dependent on the presence of substrates on the b-side since inhibition ocurred also if the b-perfusate contained no substrate and Isc was driven by luminal butyrate. Also SITS (5 mmol · l^–1) exerted only a small inhibitory effect which was not specific since it was also observed with luminal butyrate. -Cyano-m-OH-cinnamate (10 mmol · l^–1) inhibited the Isc specifically whenl-lactate was the bath substrate. Probenecid (1 mmol · l^–1) had a similar yet less marked inhibitory effect. Thed-glucose uptake from the b-side was specifically inhibited by cytochalasin B at 5 · 10^–6 mol · l^–1. We conclude that the cTAL segment of the rabbit utilizesd-glucose and/or small anions such as pyruvate orl-lactate or acetate to energize salt reabsorption. The link between substrate availability and salt reabsorption is extremely close in this nephron segment. Substrate uptake occurs from the blood side. Sugar uptake can be inhibited by cytochalasin B andl-lactate uptake by probenecid and -cyano-m-OH-cinnamat. These data suggest that substrate uptake at the basolateral cell side occurs probably via carrier systems.Parts of this study have been presented at the 57th and 58th Tagung Deutsche Physiologische Gesellschaft, 17th Meeting Europ. Soc. Clin. Investigation, and XXIX Int. Congress Physiol. Sciences. This study was supported by Deutsche Forschungsgemeinschaft Gr 480/5-7     

Transport ofl-lysine by rat intestinal brush border membrane vesicles

Brush border membranes were isolated from rat jejunum by a divalent cation precipitation method.³H-l-Lysine uptake was measured by a rapid filtration technique. Uptake after prolongued incubation periods was osmotically insensitive and represented almost exclusively binding to the vesicles. Extrapolating initial linear uptake to a zero incubation time indicated no binding of the amino acid to the external membrane surface.Sodium did not significantly alter the initial uptake rate.l-Lysine transport respresents a carrier mediated uptake in the presence and absence of sodium as indicated by the transstimulation experiments. The transport mechanism operates stereospecifically and is inhibited by other basic amino acids andl-leucine andl-phenylalanine. Saturation experiments result in aK _m of 0.26 mmoles/l and aV _max of 272 pmoles/mg protein/10 s.Inside negative anion diffusion potentials and inside negative potassium diffusion potentials (valinomycin) were unable to increase the transport rate. Transmembrane pH-gradients were also unable to alter transport.Abbreviations HEPES N-2-hydroxethylpiperazine-N-ethane sulfonic acid - Tris tris(hydroxymethyl)aminomethane - EGTA ethyleneglycolbis-(-aminoethyl-ether)-N,N tetraacetic acid - MES 2-(N-morpholino)-ethanesulfonic acid     

Characterization of the VIP- and secretin-stimulated adenylate cyclase system from human lung

The response of a crude particulate adenylate cyclase preparation from surgically removed human lung to guanine nucleotides, sodium fluoride, -adrenergic agonists, prostaglandins, vasoactive intestinal peptide (VIP), secretin, and [Val⁵]secretin was investigated. The enzyme activity increased 5, 10, and 9-fold, respectively, with GTP, Gpp(NH)p, and sodium fluoride. This activity was stimulated (in the presence as well as in the absence of added GTP) byd,l-isoproterenol,l-epinephrine andl-norepinephrine, the relative potency of these agonists being compatible with the existence of -adrenoceptors of the -adrenoceptors of the ₂ subtype. Prostaglandins E₁ and E₂, but not PGF₁ and PGF₂, stimulated the enzyme, PGE₁ being at least 10 times more potent than PGE₂. The biphasic pattern of stimulation of the same adenylate cyclase activity by VIP suggested the presence of high- and low-affinity VIP receptors coupled to the enzyme. This stimulation by VIP was not inhibited by secretin-(7–27). The stimulation of adenylate cyclase by secretin and [Val⁵]secretin was also biphasic, suggesting the coexistence of high- and low-affinity secretin receptors. Secretin-(7–27) was able to inhibit completely the secretin stimulation acting through high-affinity secretin receptors but exerted no effect on the stimulation operating through low-affinity secretin receptors, which might indicate that the latter receptors were in fact VIP-preferring receptors. [Val⁵]secretin was also used to differentiate these peptide receptors, since its properties were more VIP-like than those of secretin.Abbreviations VIP vasoactive intestinal peptide - Sn secretin - cyclic AMP cyclic adenosine 3:5-monophosphate - Gpp(NH)p guanosine 5-O-(2-3-imido)triphosphate - EGTA ethylene glycol bis (2-aminoethyl ether)-N,N,N,N-tetraacetic acid     

Evidence for carrier-mediated uptake of sugars at the serosal side of lamb colon mucosa

Sugar uptake through the basolateral membrane into epithelial cells was investigated in lamb colon stripped of serosa and muscle layers. Only the antiluminal surface of the mucosa was exposed to the incubation medium. 2-Deoxy-d-glucose (2-DG) and 3-O-methyl-d-glucose (3-MG) were used as model substrates. Both sugars were taken up by a saturable process. Transport apparently occurred by facilitated diffusion. 2-DG uptake was inhibited byd-glucose and 3-MG, but not byd-galactose and -methyl-d-glucoside and 3-MG uptake was inhibited by 2-DG andd-glucose but not by -methyl-d-glucoside. Thus 2-DG, 3-MG and glucose appear to compete for a common transport mechanism.Carrier-mediated uptake of glucose through the basolateral membranes is probably important for the energy supply of colon epithelium.Supported by the Deutsche Forschungsgemeinschaft     

Examination of a purified A′ influenza virus preparation by potentiometry

Summary The potentiometric titration of a purified influenza A virus preparation revealed 100.7×10^–4 M base and 68.8 × 10^–4 M acid-binding capacity per g. of virus protein N. The titration curve was characterized by the following fourpK values:pK ₁ = 3.37;pK ₂ = 4.50;pK ₃ = 6.37, andpK ₄ = 9.75. The isoionic point was at pH 5.43.Attempt was made to identify the dissociating groups and it was found that the carboxylic groups (pK ₁ andpK ₂) may either be glutamyl or aspartyl groups, while the cationic groups are probably the imidazolium of histidine (pK ₃) and the -amino residues of lysine (pK ₄).Inaotivation of the hemagglutinating activity of the virus preparation by mild treatment with formaldehyde at pH 8.0 resulted in a simultaneous disappearence of the -amino groups of lysine (pK ₄). The same treatment at pH 9.0 resulted in the loss of all the cationic groups previously demonstrable.The possible role of the stable positive charges on the surface of the virus at physiological pH is discussed from the point of view of the physico-chemistry of the hemagglutination.     

Distribution of anti-F(ab′)2 antibodies and the 16/6 idiotype in systemic lupus erythematosus (SLE) probands and kindreds

Levels of serum anti-F(ab)₂ antibodies and expression of the 16/6 anti-DNA idiotype were studied in 103 sera from first-degree relatives of 17 systemic lupus erythematosus (SLE) kindreds. Among healthy SLE relatives, 35.9% showed anti-F(ab)₂ elevations and 24%, Id 16/6 expression. Forty-three and two-tenths percent of healthy SLE relatives with elevated anti-F(ab)₂ also showed expression of 16/6; when Id 16/6 was positive, 16 of 25 relatives (64%) showed parallel elevations of anti-F(ab)₂. However, within individual families, distribution patterns of elevated anti-F(ab)₂ and Id 16/6 often did not coincide. Affinity-isolated anti-F(ab)₂ from four members of a single SLE kindred showed relative enrichment for Id 16/6 in only two of the four individuals studied. Moreover, none of the isolated anti-F(ab)₂ antibodies within this kindred or another kindred showing 16/6 Id expression reacted directly with 16/6 Id. Our studies suggest that whereas both anti-F(ab)₂ and Id 16/6 are increased within SLE kindreds, expression of the two does not always coincide. Furthermore, anti-F(ab)₂ antibodies do not show direct reactivity with Id 16/6. A number of anti-DNA idiotypic markers may play a role in idiotypic networks among such SLE kindreds.     

Topographical distribution of the secretin- and VIP-stimulated adenylate cyclase system in the heart of five animal species

Adenylate cylase stimulation by secretin and VIP was compared to the effect of glucagon,d,l-isoproterenol, Gpp[[NH]p, and NaF in atria and ventricles from rat, guinea pig, rabbit, dog and Cynomolgus monkey. In rat ventricular membranes, secretin was a better stimulant than VIP and was as active asd,l-isoproterenol. In rat auricular membranes both peptides were inactive. In guinea pig and rabbit heart membranes (ventricular and auricular) VIP and secretin were inactive. In dog and monkey atria, VIP stimulation of adenylate cyclase was comparable to that ofd,l-isoproterenol, secretin being inactive. In dog ventricules, VIP was less efficient thand,l-isoproterenol, secretin being inactive. In monkey ventricles, by contrast, VIP was slightly more efficient thand,l-isoproterenol, secretin having a small effect only in left ventricles. The present results established a clear difference between animal species with respect to the efficacy of the peptides of the secretin/VIP family: the presence of secretin-preferring receptors in rat heart contrasted with the presence of VIP-preferring receptors in dog and monkey heart. Our results in dog and monkey hearts suggest that VIP might be a candidate for a physiological control of heart function.Abbreviations VIP vasoactive intestinal peptide - Sn secretin - Gpp[NH]p guanosine 5-O-(2-3-imido) triphosphate - EGTA ethylene glycol bis (2-aminoethyl ether)-N,N,N,N-tetraacetic acid     

New aspects of renal potassium transport

The positive contractile effect of nitric oxide (NO) donors was studied on isolated rat ventricular cardiomyocytes within a range of a positive force/frequency relationship. We determined whether the observed effect depended on cGMP. The NO donors S-nitroso-acetyl-d,l-penicillamine (SNAP) and N-{4-[1-(3-aminopropyl)-2-hydroxy-2-nitrosohydrazino]butyl}-1,3-propanediamine (spermine-NONO) increased contractile responsiveness transiently in a concentration- and frequency-dependent manner. The influence of NO donors on cGMP levels was enhanced under beating conditions. The positive contractile effect of NO donors was inhibited by adenosine 3,5-cyclic monophosphothioate Rp diastereomer (Rp-cAMPS), but not by bisindolylmaleimide. Inhibition of the soluble guanylyl cyclase (sGC) by 1H-[1,2,4]-oxadiazole-[4,3-a]-quinoxalin-1-one (ODQ) inhibited the positive contractile effect of NO donors. Direct activation of sGC by 3-(5-hydroxymethyl-2-furyl)-1-benzylindazole (YC1) or addition of 8-bromo cGMP increased cell contractility comparably to NO donors. Inhibition of G_s proteins by NF441 inhibited the positive contractile effect of NO donors. In contrast, NO donors did not potentiate the positive contractile effect of forskolin. These results demonstrate that the positive contractile effect of NO donors on rat ventricular cardiomyocytes working in a range of a positive force/frequency relationships is enhanced. It is mediated by NO-dependent stimulation of the sGC interacting with G_s proteins.     

Formation of α-l- and β-d-fucosidase in cultures ofStreptococcus mitis

Summary The formation of-l- and-d-fucosidase fromStreptococcus mitis ATCC 15909 was studied in fermentors under controlled conditions. Both-l- and-d-fucosidase appeared extracellularly at the end of the growth period. The optimal pH for synthesis of the enzymes was 7.0 and the formation of fucosidase activities was repressed at growth below 35° C. The investigated enzymes were stable in the pH range studied. The enzymes were mainly located in the cytoplasmic fraction.