The effects of arachidonic and other fatty acids on canine neutrophil metabolism

The effect of arachidonic acid on the metabolic activity and chemiluminesence of canine neutrophils was investigated to gain further insight into its role in the neutrophil metabolic burst. Arachidonic acid was found to stimulate metabolic activity and luminol-augmented chemiluminescence. The increased metabolic activity was detected by both oxygen uptake measurements and assays of hexose monophosphate shunt activity. An inhibitor of lipoxygenase and cyclooxygenase,5, 8, 11, 14-eicosatetraynoic acid prevented the hexose monophosphate shunt response to arachidonic acid. Aspirin or indomethacin, blockers of cyclooxygenase, inhibited chemiluminescence but failed to block the metabolic response to arachidonic acid. Since superoxide dismutase and 2-deoxyglucose, a blocker of glucose metabolism, inhibited the chemiluminescent response of neutrophils to arachidonic acid, it is likely that oxygen radicals produced via the hexose monophosphate shunt are required for the chemiluminescent reaction. In addition it was found that inhibition of cyclooxygenase activity blocked chemiluminescence but not the metabolic stimulation induced by sodium fluoride, suggesting that the chemiluminescence stimulated by sodium fluoride is associated with endogenous fatty acid stores. From these studies it can be concluded that arachidonic acid products of the cyclooxygenase pathway do not play a significant role in the metabolic response of neutrophils when arachidonic acid or sodium fluoride is the stimulant while the lipoxygenase pathway appears to be involved. The metabolic response is not linked to the chemical reaction that causes neutrophil, chemiluminesence, although the chemiluminescent response depends on hexose monophosphate shunt activity and presumably the oxygen radicals that ultimately result from that process.     

Aldosterone induction of electrogenic sodium transport in the apical membrane vesicles of rat distal colon

Na-H exchange is present in apical membrane vesicles (AMV) isolated from distal colon of normal rats. Because in intact tissue aldosterone both induces amiloride-sensitive electrogenic sodium transport and inhibits electroneutral sodium absorption, these studies with AMV were designed to establish the effect of aldosterone on sodium transport. An outward-directed proton gradient stimulated 22Na uptake in AMV isolated from distal colon of normal and dietary sodium depleted (with elevated aldosterone levels) experimental rats. Unlike normal AMV, proton gradient-dependent 22Na uptake in experimental AMV was inhibited when uptake was measured under voltage-clamped conditions. 10 microM amiloride inhibited the initial rate of proton gradient-dependent 22Na uptake in AMV of normal and experimental rats by 30 and 75%, respectively. In contrast, 1 mM amiloride produced comparable inhibition (90 and 80%) of 22Na uptake in normal and experimental AMV. Intravesicular-negative potential stimulated 22Na uptake in experimental but not in normal AMV. This increase was inhibited by 90% by 10 microM amiloride. An analogue of amiloride, 5-(N-ethylisopropyl) amiloride (1 microM), a potent inhibitor of electroneutral Na-H exchange in AMV of normal rat distal colon, did not alter potassium diffusion potential-dependent 22Na uptake. Increasing sodium concentration saturated proton gradient-dependent 22Na uptake in normal AMV. However, in experimental AMV, 22Na uptake stimulated by both proton gradient and potassium diffusion potential did not saturate as a function of increasing sodium concentration. We conclude from these results that an electrically sensitive conductive channel, not electroneutral Na-H exchange, mediates 22Na uptake in AMV isolated from the distal colon of aldosterone rats.     

Carbohydrate metabolism in the rat peritoneal macrophages

Rat peritoneal macrophages derive energy differently from other tissues. Resting rat peritoneal macrophages have been taken for the present investigation. Lactate produced by extracellular glycolysis in the peritoneal lavage fluid, is readily converted into pyruvate by resting peritoneal macrophages and is oxidised in mitochondria. Glycolytic enzymes other than phosphoglucoisomerase and lactate dehydrogenase could not be substantially demonstrated. Glucose-6-phosphate dehydrogenase was detected. The presence of glucose-6-phosphate dehydrogenase along with phosphoglucoisomerase indicates the operation of the hexose monophosphate shunt as a pathway supplementary to glycolysis. Resting rat peritoneal macrophages thus appear to utilize extracellular lactate as their main energy source instead of glucose, bypass glycolysis and have active hexose monophosphate shunt.     

Mixed function oxidation of hexobarbital and generation of NADPH by the hexose monophosphate shunt in isolated rat liver cells.

Mixed function oxidation of hexobarbital and the generation of NADPH by the hexose monophosphate shunt were studied in isolated rat liver parenchymal cells from phenobarbital-pretreated and untreated animals. In cells isolated from untreated rats, a maximal rate of hexobarbital oxidation of 17 μmol·g^?1 liver wet weight·(60 min)^?1 was observed, while in cells isolated from phenobarbital-pretreated rats a maximal rate of 29 μmol·g^?1 liver wet weight·(60 min)^?1 has been obtained. On the basis of the specific radioactivity at carbon atom 1 of glucose 6-phosphate, fructose 6-phosphate and 6-phosphogluconate, determined by enzymatic decarboxylation, a ratio between NADPH formation via the hexose monophosphate shunt and NADH utilization for hexobarbital oxidation of 6:1 in untreated and 9.5:1 in pretreated cells has been obtained. With phenazine methosulfate the stimulation of NADPH generation via the hexose monophosphate shunt exceeded that observed in the presence of hexobarbital by 329 and 160%, respectively, indicating that the capacity of this pathway is sufficient to provide more reducing equivalents than are required for maximal rates of mixed function oxidation.     

Hexose monophosphate pathway in synapses

Abstract— Synaptosomes isolated from rat cerebral cortex converted [l-¹⁴C]glucose more rapidly than [6-²⁴C]glucose to ^,14CO₂. The ratio of C-l: C-6 in ¹⁴CO₂ was 3-9, thus suggesting that the hexose monophosphate shunt (HMP) pathway was functional in synapses in vitro. When changes in the ratio of C-l: C-6 in ¹⁴CO₂ were used as an index of shunt activity, glucose oxidation by this route was stimulated by electron acceptors as well as by neurohormones, including norepinephrine, acetylcholine and serotonin. Brain mince also exhibited a C-l: C-6 ratio of 3-2 when short (15 min) incubations were employed. Negative results previously reported are attributable to prolonged incubation during which depletion of NADP or randomization of the labelled carbons in radioactive glucose could have occurred. Our experiments excluded the incorporation of glucose into macromolecules as a specific role for the hexose monophosphate pathway. The generation of NADPH for numerous metabolic reactions including the maintenance of membrane SH groups and the oxidation and hydroxylation reactions may represent the functions of the hexose monophosphate in synaptosomes and account for its stimulation by neurohormones.     

Characterization and cellular localization of the epithelial Na+ channel. Studies using an anti-Na+ channel antibody raised by an antiidiotypic route.

Amiloride-sensitive Na+ channels are expressed at the apical membrane of high resistance, Na+-transporting epithelial. The specific interaction of amiloride with this transport protein suggested the feasibility of raising anti-Na+ channel antibodies by an antiidiotypic approach designed to generate antibodies directed against the amiloride-binding domain on the channel. Antiidiotypic monoclonal antibody RA6.3 mimicked the effect of amiloride by inhibiting Na+ transport across A6 cell monolayers when applied to the apical cell surface. Inhibition of transport required pretreatment of the apical cell surface with trypsin in the presence of amiloride in order to enhance accessibility of the antibody to the amiloride-binding site. This antibody specifically immunoprecipitated a large 750,000-700,000 Da protein from [35S]methionine-labeled A6 cell cultures, which was resolved further under reducing conditions as a set of polypeptides with apparent molecular masses of 260,000-230,000, 180,000, 140,000-110,000, and 70,000 Da. The antibody recognized the 140,000-Da subunit, known to contain the amiloride-binding domain, on immunoblots of purified A6 cell Na+ channel. Immunoprecipitation of apical or basolateral plasma membrane proteins selectively labeled with 125I demonstrated that expression of the oligomeric Na+ channel was restricted to the apical plasma membrane. Immunocytochemical localization in A6 cultures revealed apical membrane as well as cytosolic immunoreactive sites. Immunostaining was also observed at or near the basolateral plasma membrane.     

Sodium transport effects on the basolateral membrane in toad urinary bladder

In toad urinary bladder epithelium, inhibition of Na transport with amiloride causes a decrease in the apical (Vmc) and basolateral (Vcs) membrane potentials. In addition to increasing apical membrane resistance (Ra), amiloride also causes an increase in basolateral membrane resistance (Rb), with a time course such that Ra/Rb does not change for 1-2 min. At longer times after amiloride (3-4 min), Ra/Rb rises from its control values to its amiloride steady state values through a secondary decrease in Rb. Analysis of an equivalent electrical circuit of the epithelium shows that the depolarization of Vcs is due to a decrease in basolateral electromotive force (Vb). To see of the changes in Vcs and Rb are correlated with a decrease in Na transport, external current (Ie) was used to clamp Vmc to zero, and the effects of amiloride on the portion of Ie that takes the transcellular pathway were determined. In these studies, Vcs also depolarized, which suggests that the decrease in Vb was due to a decrease in the current output of a rheogenic Na pump. Thus, the basolateral membrane does not behave like an ohmic resistor. In contrast, when transport is inhibited during basolateral membrane voltage clamping, the apical membrane voltage changes are those predicted for a simple, passive (i.e., ohmic) element.     

Hexose monophosphate shunt,the role of its metabolites and associated disorders: A review

The hexose monophosphate (HMP) shunt acts as an essential component of cellular metabolism in maintaining carbon homeostasis. The HMP shunt comprises two phases viz. oxidative and nonoxidative, which provide different intermediates for the synthesis of biomolecules like nucleotides, DNA, RNA, amino acids, and so forth; reducing molecules for anabolism and detoxifying the reactive oxygen species during oxidative stress. The HMP shunt is significantly important in the liver, adipose tissue, erythrocytes, adrenal glands, lactating mammary glands and testes. We have researched the articles related to the HMP pathway, its metabolites and disorders related to its metabolic abnormalities. The literature for this paper was taken typically from a personal database, the Cochrane database of systemic reviews, PubMed publications, biochemistry textbooks, and electronic journals uptil date on the hexose monophosphate shunt. The HMP shunt is a tightly controlled metabolic pathway, which is also interconnected with other metabolic pathways in the body like glycolysis, gluconeogenesis, and glucuronic acid depending upon the metabolic needs of the body and depending upon the biochemical demand. The HMP shunt plays a significant role in NADPH₂ formation and in pentose sugars that are biosynthetic precursors of nucleic acids and amino acids. Cells can be protected from highly reactive oxygen species by NADPH ₂. Deficiency in the hexose monophosphate pathway is linked to numerous disorders. Furthermore, it was also reported that this metabolic pathway could act as a therapeutic target to treat different types of cancers, so treatments at the molecular level could be planned by limiting the synthesis of biomolecules required for proliferating cells provided by the HMP shunt, hence, more experiments still could be carried out to find additional discoveries.     

Mechanism of inhibition by lithium of sodium transport in the toad bladder

Lithium transport across the urinary bladder of Bufo marinus has been studied by means of the short-circuit current technique, as well as unidirectional ion flux measurements. Exposure to lithium of the epithelial (mucosal) surface of this preparation led to a slow, progressive decrease of ion transport, with increasing discrepancy between short-circuit current and lithium influx; in fact there was still an appreciable lithium influx across bladder exposed to amiloride even though short-circuit current was suppressed. Ohmic conductance and sodium efflux barely increased under these circumstances. Upon replacement of lithium by sodium on the epithelial side, the preparations recovered slowly indeed, and residual lithium could be detected in bladder tissue for more than 2 hr while the rate of sodium extrusion at the basal-lateral cell border was slowed down. Recovery from exposure to lithium was accelerated by vasopressin and amphotericin, both of which facilitate sodium entry at the apical border of the epithelium. Thus the lasting deleterious influence of lithium on sodium transport might result from the fact that this ion, once trapped in the cytoplasm, closes the sodium channels.     

Effects of an acetyl-coenzyme A carboxylase inhibitor and a sodium-sparing diuretic on aldosterone-stimulated sodium transport, lipid synthesis, and phospholipid fatty acid composition in the toad urinary bladder.

A correlation study of the effects of two agents, 2-methyl-2-[p-(1,2,3,4-tetrahydro-1-naphthyl)phenoxy]propionic acid (TPIA) and amiloride, on aldosterone-induced alterations in Na+ transport, lipid synthesis, and phospholipid fatty acid composition has been carried out in the toad urinary bladder. TPIA, an inhibitor of acetyl-CoA carboxylase, inhibits aldosterone-stimulated Na+ transport as well as hormone-induced lipid synthesis and the increase in weight percentage of phospholipid long-chain polyunsaturated fatty acids. Amiloride, a diuretic which blocks sodium entry into the transporting epithelium, does not alter aldosterone's effects on lipid and fatty acid metabolism but prevents the hormone-induced increase in Na+ transport. These results support the conclusion that aldosterone increases Na+ transport in the toad urinary bladder by altering membrane fatty acid metabolism and that the lipid biosynthetic events following aldosterone treatment are a primary response to the hormone and not secondary to increased Na+ transport.     

Amiloride-sensitive trypsinization of apical sodium channels. Analysis of hormonal regulation of sodium transport in toad bladder

Incubation of the mucosal surface of the toad urinary bladder with trypsin (1 mg/ml) irreversibly decreased the short-circuit current to 50% of the initial value. This decrease was accompanied by a proportionate decrease in apical Na permeability, estimated from the change in amiloride-sensitive resistance in depolarized preparations. In contrast, the paracellular resistance was unaffected by trypsinization. Amiloride, a specific blocker of the apical Na channels, prevented inactivation by trypsin. Inhibition of Na transport by substitution of mucosal Na, however, had no effect on the response to trypsin. Trypsinization of the apical membrane was also used to study regulation of Na transport by anti-diuretic hormone (ADH) and aldosterone. Prior exposure of the apical surface to trypsin did not reduce the response to ADH, which indicates that the ADH-induced Na channels were inaccessible to trypsin before addition of the hormone. On the other hand, stimulation of short-circuit current by aldosterone or pyruvate (added to substrate-depleted, aldosterone-repleted bladders) was substantially reduced by prior trypsinization of the apical surface. Thus, the increase in apical Na permeability elicited by aldosterone or substrate involves activation of Na channels that are continuously present in the apical membrane in nonconductive but trypsin-sensitive forms.     

Metabolism of glucose into glutamate via the hexose monophosphate shunt and its inhibition by 6-aminonicotinamide in rat brain in vivo

The treatment of rats for 4 h with 6-aminonicotinamide (60 mg kg-1) resulted in an 180-fold increase in the concentration of 6-phosphogluconate in their brains; glucose increased 2.6-fold and glucose 6-phosphate, 1.7-fold. Moreover, lactate decreased by 20%, glutamate by 8% and gamma-aminobutyrate by 12%, and aspartate increased by 10%. No significant changes were found in glutamine and citrate. In blood, 6-phosphogluconate increased 5-fold; glucose, 1.4-fold and glucose 6-phosphate, 1.8-fold. The metabolism of glucose in the rat brain, via both the Embden-Meyerhof pathway and the hexose monophosphate shunt, was investigated by injecting [U-14C]glucose or [2-14C]glucose, and that via the hexose monophosphate shunt alone by injecting [3,4-14C]glucose. The total radioactive yield of amino acids in the rat brain was 5.63 mumol at 20 min after injection of [U-14C]glucose, or 5.82 mumol after injection of [2-14C]glucose; by contrast, it was 0.62 mumol after injection of [3,4-14C]glucose. The treatment of rats with 6-aminonicotinamide showed significant decreases in these values, owing to decreases in the radioactive yields of glutamate, glutamine, aspartate, gamma-aminobutyrate, and alanine+glycine+serine. Glutamate isolated from the brain contained approximately 43% of its radioactivity in carbon 1 after injection of [3,4-14C]glucose, in contrast to 13% and 18% after injection of [U-14C]glucose and [2-14C]glucose, respectively, in both the control and treated rats. The calculations based on these findings showed that approximately 69% of the 14C-labelled glutamate was formed from [14C]acetyl coenzyme A (acetyl CoA) and the residual 31% by 14CO2 fixation of pyruvate after injection of [3,4-14C]glucose in both control and treated rats. The results gave direct evidence that glutamate and gamma-aminobutyrate in the brain were formed by metabolism of glucose via the hexose monophosphate shunt as well as via the Embden-Meyerhof pathway. From the radioactive yields of glutamate formed via [14C]acetyl CoA it was estimated that approximately 7.8% of the total glucose utilized was channelled via the hexose monophosphate shunt. Assuming that [14C]glutamate formed by carbon-dioxide fixation of pyruvate was also dependent on the metabolism of glucose through the hexose monophosphate shunt, the estimated value was approximately 9.5% of the total glucose converted into glutamate. The results of the present investigation, taken in conjunction with other findings, suggest that the utilization of glucose via the hexose monophosphate shunt is functionally important in the rat brain.     

Glutaraldehyde fixation of sodium transport in dog red blood cells

The large increase in passive Na flux that occurs when dog red blood cells are caused to shrink is amiloride sensitive and inhibited when Cl is replaced by nitrate or thiocyanate. Activation and deactivation of this transport pathway by manipulation of cell volume is reversible. Brief treatment of the cells with 0.01-0.03% glutaraldehyde can cause the shrinkage-activated transporter to become irreversibly activated or inactivated, depending on the volume of the cells at the time of glutaraldehyde exposure. Thus, if glutaraldehyde is applied when the cells are shrunken, the amiloride-sensitive Na transporter is activated and remains so regardless of subsequent alterations in cell volume. If the fixative is applied to swollen cells, no amount of subsequent shrinkage will turn on the Na pathway. In its fixed state, the activated transporter is fully amiloride sensitive, but it is no longer inhibited when Cl is replaced by thiocyanate. The action of glutaraldehyde thus allows one to dissect the response to cell shrinkage into two phases. Activation of the pathway is affected by anions and is not prevented by amiloride. Once activated and fixed, the anion requirement disappears. Amiloride inhibits movement of Na through the activated transporter. These experiments demonstrate how a chemical cross-linking agent may be used to study the functional properties of a regulable transport pathway.     

Nitric Oxide Links the Apical Na+ Transport to the Basolateral K+ Conductance in the Rat Cortical Collecting Duct

We have used the patch clamp technique to study the effects of inhibiting the apical Na⁺ transport on the basolateral small-conductance K⁺ channel (SK) in cell-attached patches in cortical collecting duct (CCD) of the rat kidney. Application of 50 μM amiloride decreased the activity of SK, defined as nP _o (a product of channel open probability and channel number), to 61% of the control value. Application of 1 μM benzamil, a specific Na⁺ channel blocker, mimicked the effects of amiloride and decreased the activity of the SK to 62% of the control value. In addition, benzamil reduced intracellular Na⁺ concentration from 15 to 11 mM. The effect of amiloride was not the result of a decrease in intracellular pH, since addition 50 μM 5-(n-ethyl-n-isopropyl) amiloride (EIPA), an agent that specifically blocks the Na/H exchanger, did not alter the channel activity. The inhibitory effect of amiloride depends on extracellular Ca²⁺ because removal of Ca²⁺ from the bath abolished the effect. Using Fura-2 AM to measure the intracellular Ca²⁺, we observed that amiloride and benzamil significantly decreased intracellular Ca²⁺ in the Ca²⁺-containing solution but had no effect in a Ca²⁺-free bath. Furthermore, raising intracellular Ca²⁺ from 10 to 50 and 100 nM with ionomycin increased the activity of the SK in cell-attached patches but not in excised patches, suggesting that changes in intracellular Ca²⁺ are responsible for the effects on SK activity of inhibition of the Na⁺ transport. Since the neuronal form of nitric oxide synthase (nNOS) is expressed in the CCD and the function of the nNOS is Ca²⁺ dependent, we examined whether the effects of amiloride or benzamil were mediated by the NO-cGMP–dependent pathways. Addition of 10 μM S-nitroso-n-acetyl-penicillamine (SNAP) or 100 μM 8-bromoguanosine 3′:5′-cyclic monophosphate (8Br-cGMP) completely restored channel activity when it had been decreased by either amiloride or benzamil. Finally, addition of SNAP caused a significant increase in channel activity in the Ca²⁺-free bath solution. We conclude that Ca²⁺-dependent NO generation mediates the effect of inhibiting the apical Na⁺ transport on the basolateral SK in the rat CCD.     

Effects of cadmium on Na+ transport in the isolated skin of the toad Pleurodema thaul

Cadmium ions applied to either (outer or inner) surface of the isolated toad skin dose-dependently increased the short-circuit current (SCC), the potential difference (V) and the active sodium conductance (G(Na)) in the concentration range 0.07-0.50mM. Maximal stimulatory effect was over 30% with an EC(50) of about 0.1mM. The effect of the highest concentration used (0.75mM) decreased considerably, and when it was applied to the inner surface (10 experiments), induced between 30% and 40% inhibition of the electric parameters in four experiments. Pretreatment with amiloride inverted the stimulatory effect of externally applied Cd(2+), suggesting competitive action on the apical Na(+) channel. The effect of noradrenaline (NA) was increased after outer application of Cd(2+) and decreased after inner application of the metal: the latter effect might be due to cadmium inhibition of the activity of Na(+),K(+)-ATPase. On the other hand, pretreatment with amiloride was followed by partial although transient reversal of its effects by serosal Cd(2+), which might be explained by action of cadmium on cytoplasmic lysine residues concerned with Na(+) channel gating. The amiloride test showed that the increment of the electric parameters was due principally to stimulation of the driving potential for Na(+) (V-E(Na(+))) and that inhibition was accompanied by a reduction in the V-E(Na(+)) and by a significant decrease in skin resistance indicating possible disruption of membrane or cell integrity. These data are in favor of the possibility that externally applied Cd(2+) activates toad skin ion transport, partly by increasing apical sodium conductance and also by stimulating the V-E(Na(+)), and that internally applied Cd(2+), with easier access to membrane and cellular constituents, may inhibit the sodium pump.     

Transport-related modulation of the membrane properties of toad urinary bladder epithelium.

Impedance analysis and transepithelial electrical measurements were used to assess the effects of the apical membrane Na+ channel blocker amiloride and anion replacement on the apical and basolateral membrane conductances and areas of the toad urinary bladder (Bufo marinus). Mucosal amiloride addition decreased both apical and basolateral membrane conductances (Ga and Gbl, respectively) with no change in membrane capacitances (Ca and Cbl). Consequently, the specific conductances of these membranes decreased without significant changes in membrane area. Following amiloride removal, an increase was obtained in the steady-state rate of sodium transport compared to values before amiloride addition. This increase was independent of the initial transport rate, suggesting activation of a quiescent pool of apical sodium channels. Chloride replacement by acetate or gluconate had no significant effects on apical or basolateral membrane capacitances. The effects of these replacements on membrane conductances depended on the anion species. Gluconate (which induces cell shrinkage) decreased both membrane conductances. In contrast, acetate (which induces cell swelling) increased Ga and had no effect on Gbl. The increase in the apical membrane conductance was due to an increase in the amiloride-sensitive Na+ conductance of this membrane. In summary, mucosal amiloride addition or chloride replacements led to changes in membrane conductances without significant effects on net membrane areas.     

Aldosterone upregulates purinergic responses in larval amphibian skin epithelium

Bullfrog tadpoles respond to apical application of 100 microM amiloride, acetylcholine (ACh) or ATP with a sharp transient inward (apical to basolateral) cation current. In adult skin, amiloride blockable transepithelial Na+ transport is upregulated by the hormone aldosterone. Tadpoles were treated in vivo with aldosterone and changes in short circuit current (Isc) in response to apical application of ATP were determined. Bullfrog tadpoles were exposed to aldosterone (10(-6) M) for periods ranging from 3 h to 60 h. Skins from 60-h aldosterone-treated animals showed a two- to three-fold increase in apical ATP-activated short circuit current when compared to animals treated with vehicle alone. Sodium replacement with a large, nonpermeable cation resulted in no measurable increase in Isc after exposure to ligand, consistent with ATP activation of an inward cation current and not chloride efflux. Activation/desensitization time courses and treatment with blockers revealed no measurable differences between aldosterone-treated and non-treated skins. Activation by amiloride and ACh gave essentially identical results. Studies with RT/PCR showed significant increases over controls of levels of mRNA associated with P2X channels. Given these data, our working hypothesis is that all three ligands activate the same process that exhibits both purinergic and cholinergic characteristics. These data are consistent with aldosterone upregulation of ATP gated channels expressed in the apical membrane of larval frog skin.     

Stimulation of sodium transport by aldosterone and arginine vasotocin in A6 cells

The effects of aldosterone and arginine vasotocin (AVT) on transepithelial Na+ transport of cultured A6 cells were investigated. All experiments were performed with cells grown on Millicell TM culture-plate inserts for a period of 2-4 weeks in defined, serum-free medium. Omitting fetal bovine serum 2 days after seeding the cells on filters did not influence potential difference (PD) development or the hormonal responses tested. The cell layers were placed in an Ussing chamber for short-circuit current (ISC) and transepithelial conductance (G) measurements. Base-line values were (n = 93): PD, 51.0 +/- 0.2 mV (apical side negative); ISC, 14.55 +/- 0.06 microA/cm2; G, 0.306 +/- 0.001 mS/cm2. ISC and G were higher in cells pretreated with 10(-7) M aldosterone for 24 h in the incubator, when compared to controls (ISC, 28 +/- 2 vs. 16 +/- 2 microA/cm2, G, 0.41 +/- 0.04 vs. 0.26 +/- 0.01 mS/cm2, n = 5) and both remained stable for at least 6 h. In cells not treated with aldosterone, 10(-7) M AVT increased ISC within 1 min after addition, producing a maximum ISC within 15 min which then declined to baseline levels over the next 5 h. Addition of AVT to aldosterone-pretreated cells resulted in a significantly greater peak increase in ISC than in non-pretreated cells (change in ISC compared to controls: 8.1 +/- 0.4 vs. 4.9 +/- 0.4 microA/cm2, n = 5, P less than 0.001), indicating a synergistic effect. A dose-response curve for amiloride obtained in the presence of AVT showed that amiloride completely inhibits ISC. Pretreatment of the A6 cells with aldosterone for 24 h shifted the amiloride dose-response curve to the right, as expressed in a doubling of the apparent Ki value (from 0.17 +/- 0.02 to 0.33 +/- 0.04 microM). In conclusion, A6 cells grown in defined, serum-free medium express a greater than additive synergism between aldosterone and AVT in stimulating transepithelial Na+ transport.     

Effect of the amputation of the cotyledon and of the application of growth regulators on the transport of32P in decapitated pea seedlings

When the epicotyl and one cotyledon is cut off from pea seedlings, only the axillary of the amputated cotyledon is known to grow. When³²P is applied to the roots of such plants, then a higher radioactivity appears in the axillary of the amputated cotyledon already 24 hrs. after amputation of one cotyledon, although this axillary is of the same size at this time as that of the remaining cotyledon. This fact indicates a more extensive material transport to the axillary bud of the amputated cotyledon already during the first day after amputation The effect of individual regulators on the³²P transport was investigated in an experiment where pea seedlings cultivated in the dark were decapitated and a 0.5% paste, containing the regulatory compounds was placed either on the cutting surface in the apical part of the epicotyl stump or in its central part. After a week the plant roots were supplied with³²P and its transport to the upper part of the epicotyl stump was followed. This transport increased about 10-fold in the case of a paste, containing indolyl acetic acid, when the paste was spread on the apical cutting surface of the stump. However, the transport was inhibited when the paste was applied in the central part of the stump. These results indicate that only the transport of³²P towards the paste with indolyl acetic acid is accelerated, whereas it is decelerated above this paste. A paste, containing triodobenzoic acid inhibited³²P transport only when applied to the apical cutting surface of the epicotyl stump and not when spread over the middle part. In this case³²P transport was more rapid above the paste than towards the paste. The situation was similar in the case of gibberellin and kinetin.     

Glycolysis in the eye tissues of the rabbit in ontogeny. I. The enzymes of glycolysis and hexosemonophosphate shunt]

The activity of the enzymes of glycolysis (phosphofructokinase, aldolase, pyruvate kinase, lactate dehydrogenase) and hexose monophosphate shunt (glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase) was determined in the eye tissues of the rabbit at different stages of ontogenesis. The activity of these enzymes in the retina was shown to be higher than in other eye tissues. In the uveal tract (iris, ciliary bodies, uvea) the activity of glycolytic enzymes changes with the age. The greatest changes in the activity of enzymes were found during the period of the opening of eyelids. The activity of the enzymes of hexose monophosphate shunt in the eye tissues increases with the age. The relative activity of dehydrogenases of the hexose monophosphate shunt after the establishment of visual function is, however, not high and does not exceed that of phosphofructokinase and pyruvate kinase in the eye tissues of the rabbit.