Purification and properties of 5'-nucleotidase from the membrane of Vibrio costicola, a moderately halophilic bacterium.

Two different Mg2+-dependent adenosine 5'-triphosphate-hydrolyzing activities were detected in membranes of Vibrio costicola, a novel 5'-nucleotidase and an N,N'-dicyclohexylcarbodiimide-sensitive adenosine triphosphatase. The former and the latter had different requirements for Mg2+ and were selectively assayed in the membranes by using, respectively, 20 and 2 mM Mg2+. The two enzymes were extracted with a combination of Triton X-100 and octylglucoside, separated on a diethylaminoethyl cellulose column, and purified on glycerol gradients. The purified 5'-nucleotidase consisted of one major polypeptide of 70,000 daltons when analyzed on polyacrylamide gels in the presence of sodium dodecyl sulfate. The purified 5'-nucleotidase was similar in substrate specificities, divalent cation specificities, and pH profiles to the membrane-bound N,N'-dicyclohexylcarbodiimide-insensitive nucleotide-phosphohydrolyzing activity. The enzyme hydrolyzed nucleoside 5'-tri, 5'-di, and 5'-monophosphates at comparable rates. Inorganic pyrophosphate, p-nitrophenyl phosphate, glucose 6-phosphate, beta-glycerophosphate, adenosine 5'-diphosphate glucose, adenosine 3'-monophosphate, and cyclic adenosine 3',5'-monophosphate were not hydrolyzed, either im membranes or by the purified 5'-nucleotides. Actions of NaCl and KCl on the activity of the 5'-nucleotidase were studied. The enzyme was activated by both NaCl and KCl; the activation profiles however, were different for the membrane-bound and purified 5'-nucleotidase. The purified enzyme, unlike the membrane-bound enzyme, was markedly stimulated by high concentrations of NaCl (up to 3 M).     

Inhibition of sodium-independent amino acid transport by dexamethasone in rat hepatoma cells

We studied the uptake of leucine, phenylalanine, and the amino acid analog, 2-aminonorborane-2-carboxylic acid, by rat hepatoma cells in tissue culture. The uptake of these amino acids was partially mediated by a plasma membrane transport system similar to the L agency described in other cell types in that it does not require extracellular sodium and is subject to trans-stimulation. Initial rates of sodium-independent transport of these amino acids were calculated using mathematical transformations of the uptake time course curves. The glucocorticoid dexamethasone inhibits the activity of this transport system; the initial rates of sodium-independent uptake of leucine, phenylalanine, and 2-aminonorborane-2-carboxylic acid are decreased by approximately one-third (average = 30%, n = 19) after incubation of HTC cells with 0.1 microM dexamethasone. This inhibition requires at least 15 h, reaching a maximum at 24 h of exposure of the cells to the hormone. Dexamethasone has an asymmetrical effect on sodium-independent amino acid transport in that exposure of the cells to the hormone does not inhibit the rates of outflow of leucine or phenylalanine from preloaded cells into medium without sodium. Inhibition of uptake is blocked by 0.1 mM cycloheximide and 4 microM actinomycin D, indicating the need for continuous protein synthesis for dexamethasone action. Insulin, which is known to partially reverse the inhibitory effect of dexamethasone on the A amino acid transport system in HTC cells, does not alter the action of dexamethasone on the L system. Previous investigations have demonstrated inhibition by dexamethasone of at least two distinct sodium-dependent amino acid transport activities in HTC cells. The data presented here, showing inhibition by the glucocorticoid of a sodium-independent transport activity, indicate that the effect of the hormone is independent of the energy source of the amino acid transport systems affected.     

Glutamine transport by rat basolateral membrane vesicles

Glutamine, a neutral amino acid, is unlike most amino acids, has two amine moieties which underlies its importance as a nitrogen transporter and a carrier of ammonia from the periphery to visceral organs. The gastrointestinal tract utilizes glutamine as a respiratory substrate. The intestinal tract receives glutamine from the luminal side and from the arterial side through the basolateral membranes of the enterocyte. This study characterizes the transport of glutamine by basolateral membrane vesicles of the rat. Basolateral membranes were prepared by a well validated technique of separation on a percoll density gradient. Membrane preparations were enriched with Na+/K+-ATPase and showed no 'overshoot' phenomena with glucose under sodium-gradient conditions. Glutamine uptake represented transport into the intravesicular space as evident by an osmolality study. Glutamine uptake was temperature sensitive and driven by an inwardly directed sodium gradient as evident by transient accumulation of glutamine above the equilibrium values. Kinetics of glutamine uptake under both sodium and potassium gradients at glutamine concentrations between 0.01 and 0.6 mM showed saturable processes with Vmax of 0.39 +/- 0.008 and 0.34 +/- 0.05 nmol/mg protein per 15 s for both sodium-dependent and sodium-independent processes, respectively. Km values were 0.2 +/- 0.01 and 0.55 +/- 0.01 mM, respectively. pH optimum for glutamine uptake was 7.5. Imposition of negative membrane potential by valinomycin and anion substitution studies enhanced the sodium-dependent uptake of glutamine suggesting an electrogenic process, whereas the sodium-independent uptake was not enhanced suggesting an electroneutral process. Other neutral amino acids inhibited the initial uptake of glutamine under both sodium-dependent and sodium-independent conditions. We conclude that glutamine uptake by basolateral membranes occurs by carrier-mediated sodium-dependent and sodium-independent processes. Both processes exhibit saturation kinetics and are inhibited by neutral amino acids. The sodium-dependent pathway is electrogenic whereas the sodium-independent pathway is electroneutral.     

Effect of phosphate esters, nucleotides and nucleosides on 5'-nucleotidase of cultured mouse macrophages.

Mouse peritoneal macrophages elicited by intraperitoneal injection of sodium caseinate exhibit low levels of ecto-5'-nucleotidase (E. C. 3.1.3.5) activity in contrast to macrophages obtained by peritoneal lavage. When elicited cells were cultured under standard conditions in the presence of serum, a 2.5-fold increase in 5'-nucleotidase activity was observed over a period of 48 hours. Addition of adenosine monophosphate to the culture medium led to an augmented (5-fold) increase in the specific activity (per unit cell protein) as well as an absolute increase (per culture plate) of 5'-nucleotidase. Other adenosine-containing compounds also had stimulatory effects. The levels of this enzyme thus appear to be regulated by the extracellular levels of adenosine nucleotides. The product of the enzymatic reaction--adenosine--when added to the medium exhibited a toxic effect on these cells--as did adenosine monophosphate. However, the former substance did not augment the increase in enzyme activity during culture. The toxic effect could be suppressed when the cells were cultured in the presence of uridine 5'-monophosphate. The latter substance also depressed the stimulation of enzyme activity due to AMP.     

Inhibitors of prostaglandin synthesis block the induction of staphylococcal enterotoxin B-activated T-suppressor cells

A variety of arachidonic acid metabolites possess the ability to modulate immune cell function. Various inhibitors of arachidonic acid metabolism were compared with regard to their effects on T-suppressor (Ts) cell function. Using staphylococcal enterotoxin B (SEB) to activate Lyt-2+ Ts cells, it was shown that indomethacin and 5,8,11,14-eicosatetraynoic acid (ETYA) inhibit the induction phase, but not the expression phase, of suppressor cell activity. Agents which inhibit thromboxane synthetase or lipoxygenase activities (imidazole, nordihydroguaiaretic acid, and pyrogallol) were not found to affect Ts cell induction. Since inhibitors of prostaglandin synthesis are thought to induce lower levels of cyclic adenosine monophosphate, an attempt to overcome the indomethacin inhibition of Ts cell induction by modulating cyclic adenosine monophosphate levels was made. It was found that theophylline and isoproterenol are not able to overcome the inhibition by indomethacin of Ts cell activity. These results strongly suggest that induction of Ts cells by SEB is dependent on the synthesis of products of the prostaglandin synthetase pathway.     

Further characterization of adenosine transport in renal brush-border membranes

Adenosine transport has been further characterized in rat renal brush-border membranes (BBM). The uptake shows two components, one sodium-independent and one sodium-dependent. Both components reflect, at least partly, translocation via a carrier mechanism, since the presence of adenosine inside the vesicles stimulates adenosine uptake in the presence as well as in the absence of sodium outside the vesicles. The sodium-dependent component is saturable (Km adenosine = 2.9 microM, Vmax = 142 pmol/min per mg protein) and is abolished at low temperatures. The sodium-independent uptake has apparently two components: one saturable (Km = 4-10 microM, Vmax = 174 pmol/min per mg protein) and one non-saturable (Vmax = 3.4 pmol/min per mg protein, Km greater than 2000 microM). Inosine, guanosine, 2-chloroadenosine and 2'-deoxyadenosine inhibit the sodium-dependent and -independent transport, as shown by trans-stimulation experiments, probably because of translocation via the respective transporter. Uridine and dipyridamole inhibited only the sodium-dependent uptake. Other analogs of adenosine showed no inhibition. The kinetic parameters of the inhibitors of the sodium-dependent component were further investigated. Inosine was the most potent inhibitor with a Ki (1.9 microM) less than the Km of adenosine. This suggests a physiological role for the BBM ecto-adenosine deaminase (enzyme which extracellularly converts adenosine to inosine), balancing the amount of nucleoside taken up as adenosine or inosine by the renal proximal tubule cell.     

Different effects of cGMP and cAMP in the intestine of the European eel,Anguilla anguilla

The regulation of salt absorption in the sea water cell intestine was studied by evaluating the effects of theophylline, 8 Br cyclic adenosine monophosphate, 8 Br cyclic guanosine monophosphate, atriopeptin III, porcine vasoactive intestinal peptide and prostaglandin E ₁ on the short-circuit current, the transepithelial voltage difference and conductance and on the dilution potentials. It was shown that theophylline increased the transepithelial conductance and reduced the magnitude of the dilution potentials, indicating that the drug increase the anion conductance of the tight junctions. In addition its inhibitory effect on short-circuit current and transepithelial voltage difference suggests that theophylline also affects the transcellular transport mechanisms. It was shown that 8 Br cyclic guanosine monophosphate and 8 Br cyclic adenosine monophosphate affect transcellular mechanisms underlying Cl^– transport since both compounds reduced short-circuit current and transepithelial voltage difference; however, cyclic adenosine monophosphate is less effective since unlike cyclic guanosine monophosphate, even at maximal concentration, it was not able to completely abolish transepithelial voltage difference and short-circuit current. The effects of cyclic guanosine monophosphate and cyclic adenosine monophosphate were not additive even if cyclic guanosine monophosphate may produce further inhibition of ion transport in 8 Br cyclic adenosine monophosphate-treated tissues. In addition, cyclic guanosine monophosphate but not cyclic adenosine monophosphate reduced the magnitude of the dilution potentials, suggesting that cyclic guanosine monophosphate acts also on the paracellular pathway. Rat atriopeptin III, a peptide known to increase cyclic guanosine monophosphate cellular levels, behaved like 8 Br cyclic guanosine monophosphate since it lowered the dilution potentials and reduced short-circuit current and transepithelial voltage difference to near zero values, suggesting that the hormone modulates both paracellular and transcellular transport mechanisms, probably acting on the Na-K-2Cl cotransport. Agents acting via cyclic adenosine monophosphate, like porcine vasoactive intenstinal peptide and prostaglandin, behaved like 8 Br cyclic adenosine monophosphate. They were less effective in inhibiting ion transport and did not interfere with the paracellular pathway.Abbreviations AP III rat artriopeptin III - 8 Br cAMP 8 Br cyclic adenosine monophosphate - 8 Br cGMP 8 Br cyclic guanosine monophosphate - g _t transepithelial conductance - I _sc short circuit current - IC ₅₀ half-maximal inhibitory concentration - NaK ATPase Na-K-adenosine monophosphate - NPPB 5-nitro-2-(3-phenylpropylamino)-benzoic acid - PGE prostaglandin E ₁ - R _t tissue resistance - SITS 4-acetamide-4-isothiocyano-stilbene-2,2-disulfonic acid - V _t transepithelial voltage difference - VIP porcine vasoactive intestinal peptide     

Uptake of Kynurenine into Rat Brain Slices

The transport of [3H]kynurenine ([3H]KYN) into slices from rat tissue was examined in vitro. Brain accumulated KYN seven to eight times more effectively than any of several peripheral organs. Of all the organs tested, only the brain exhibited a sodium-dependent component of the uptake process. After an incubation period of 1 h, sodium-dependent transport amounted to 60% of total uptake. Both processes were abolished by prior sonication of the tissue and significantly inhibited by inclusion of metabolic blockers in the incubation medium. Time resolution showed that the sodium-independent uptake occurred rapidly and reached saturation within 30 min. In contrast, sodium-dependent transport was linear for at least 2 h of incubation. Brain regional analysis revealed a sevenfold difference between the areas of highest (cortex) and lowest (cerebellum) uptake. With the exception of cerebellar tissue, the ratio between sodium-dependent and sodium-independent processes was consistent among brain regions. Kinetic analyses were performed on striatal slices and revealed a Km of 927 microM and a Vmax of 18 nmol/h/mg of protein for the sodium-dependent process, and a Km of 3.8 mM and a Vmax of 38 nmol/10 min/mg of protein for the sodium-independent transport. The transporters were equally amenable to inhibition by KYN and tryptophan, indicating that KYN entry into the cell may be mediated by neutral amino acid uptake sites. No strict stereoselectivity existed, but L enantiomers were clearly more active than the D forms.(ABSTRACT TRUNCATED AT 250 WORDS)     

5'-Nucleotidase activity of isolated mature rat cardiac myocytes

Specific location of 5'-nucleotidase in the heart has been uncertain, some authors citing evidence for an exclusively non-myocyte location, while other data point to the existence of cytoplasmic and membrane-bound fractions. Single myocytes isolated from mature rat heart, and free of endothelial or interstitial cells, have been used to establish that muscle cells of the myocardium are rich in 5'-nucleotidase, exhibiting activity sufficient to account for the total myocardial content of this enzyme. All 5'-nucleotidase is accessible to extracellular AMP. Inhibitors of 5'-nucleotidase and adenosine transport have been used to establish that only the adenosine component of adenine nucleotides is taken up by myocytes, but hydrolysis of AMP by 5'-nucleotidase does not commit the adenosine formed to transport across the sarcolemmal membrane. Myocytes also have ecto-phosphatases which hydrolyse ADP and ATP.     

The effect of dibutyryl cyclic AMP on the uptake of taurocholic acid by isolated rat liver cells

The effect of dibutyryl cyclic AMP on the uptake of taurocholic acid by isolated rat hepatocytes was studied. In the presence of low levels (10–100 μM) of the cyclic nucleotide the initial rate of uptake was increased significantly, with a peak occurring at about 20 μM. In contrast, concentrations of dibutyryl cyclic AMP between 200 μM and 1 mM caused a significant decrease in the initial rate of uptake of the bile acid by the cells. Sodium-dependent transport of taurocholic acid was found to be enhanced by 20 μM dibutyryl cyclic AMP, but sodium-independent uptake appeared to be unaffected. Inhibition by 1 mM dibutyryl cyclic AMP, however, was found to occur in both the sodium-dependent and -independent components of the transport system. The initial rate of taurocholic acid uptake in hepatocytes incubated with 1.2 mM extracellular calcium was increased compared to that in calcium-depleted cells, and this increase was entirely due to enhanced sodium-dependent transport. 1.2 mM calcium and 20 μM dibutyryl cyclic AMP together did not stimulate the uptake rate to a greater extent either treatment alone. It is conclude that calcium and low levels of dibutyryl cyclic AMP alter the rate of taurocholic acid uptake by changing the flux of sodium in the hepatocytes. The inhibitory effect of 1 mM dibutyryl cyclic AMP was not relieved by the presence of 1.2 mM calcium in the cell incubation medium. The results show that dibutyryl cyclic AMP can affect the rate of transport of bile acid into liver cells, and suggest a possible regulatory role for cyclic AMP in this process.     

Calcitonin stimulates adenylate cyclase activity, the accumulation of cyclic adenosine 3',5' monophosphate and the release of prostaglandin E2 in rat intestinal mucosa

Calcitonin stimulates intestinal fluid and electrolyte secretion by an unclear mechanism. The present results show that synthetic salmon calcitonin significantly stimulates adenylate cyclase activity in the membranes of rat intestinal mucosal cells. The effect of the hormone was in a dose-dependent manner for a dose range of 10(-9)-10(-7) M. The stimulated enzyme activity was followed by a progressive accumulation of cyclic adenosine 3',5' monophosphate and release of prostaglandin E2 in these cells during a 20 min experimental period of time. These results are discussed, and suggest that the formation of cyclic adenosine 3',5' monophosphate possibly mediates the action of calcitonin on intestinal cell functions.     

Effect of a steroidal oral contraceptive on intestinal absorptive functions in proteins deficient rat

The effects of steroidal oral contraceptive norethynodrel plus ethinylestradiol-3-methyl ether (SOC) at a daily dose of 5 mg: 0.06 mg per kg body weight for 28 days on intestinal absorptive functions have been investigated in protein-deficient female albino rats. The administration of this contraceptive caused significant increase in glucose and amino acids uptake but had no effect on calcium and zinc uptake in pair-fed as well as in protein-deficient rat. Further studies carried out on glucose transport system showed that the transport of sodium-dependent glucose was significantly enhanced while that of sodium-independent glucose remained unaltered in drug-treated animals. Kinetic studies of glucose transport in the presence of sodium ions revealed that SOC treatment affected the rate of uptake of glucose by elevating Vmax, but the apparent Kt value remained the same in treated and untreated animals.     

Modulation of amino acid transport in preimplantation mouse embryos by low concentrations of non-ionic and zwitterionic detergents

In 4-cell embryos (but not in blastocysts), Triton X-100, a non-ionic detergent, stimulated leucine, phenylalanine, methionine and glutamic acid transport from 1.6 to 3.2-fold. All of these amino acids were transported exclusively by a sodium-independent mechanism. Triton X-100, however, did not stimulate the transport of other amino acids tested in 4-cell embryos. Furthermore, phenylalanine transport rates were stimulated about 2-fold at the 4-cell stage by all of the non-ionic and zwitterionic detergents tested at concentrations which were approximately one-tenth of the critical micellar concentration for each detergent. These concentrations did not block development, disrupt the cells, or make the cell membranes freely permeable. At the blastocyst stage, Z312, a zwitterionic detergent, inhibited the transport of phenylalanine and alanine and stimulated the transport of lysine, a pattern previously found to be linked to the sodium-dependent amino acid transport mechanism. We suggest that Z312 may be acting upon some component of sodium-dependent amino acid transport in blastocysts. The non-ionic and zwitterionic detergents seemed to have a common effect on amino acid transport in 4-cell embryos but elicited varied transport responses from blastocysts. These differential responses to detergents by blastocysts may reflect intrinsic changes in membrane composition and/or organization which occur during the normal course of preimplantation development.     

Fatty Acid Degradation in Escherichia coli: Requirement of Cyclic Adenosine Monophosphate and Cyclic Adenosine Monophosphate Receptor Protein for Enzyme Synthesis

The strong repression of inducible synthesis of the enzymes of fatty acid degradation by glucose can be partially relieved by the addition of cyclic adenosine 3',5' monophosphate (cyclic AMP) to the growth medium. This reversal of the glucose effect by cyclic AMP is not observed in a mutant (K29) that is unable to grow on fatty acids as sole carbon source and that was found to synthesize low levels of several enzymes specified by the fad regulon. In a revertant selected for the ability to grow on oleate these effects are concomitantly relieved. By both genetic (co-transduction of the mutation with the strA locus) and biochemical experiments (an extract of the mutant strain does not show the cyclic AMP-dependent stimulation of the deoxyribonucleic acid-directed in vitro synthesis of the enzymes of the gal operon), it is demonstrated that the mutant lacks functional cyclic AMP receptor protein (CR protein). It is concluded that, like many other inducible enzyme systems, expression of the enzymes of the fad system requires cyclic AMP and the CR protein.     

Adenosine production and its interaction with protection of ischemic and reperfusion injury of the myocardium

Adenosine exerts cardioprotective effects on the ischemic myocardium. A flexibly mounted microdialysis probe was used to measure the concentration of interstitial adenosine and to assess the activity of ecto-5'-nucleotidase (a key enzyme responsible for adenosine production) in in vivo rat hearts. The level of adenosine during perfusion of adenosine 5'-adenosine monophosphate (AMP) was given as an index of the activity of ecto-5'-nucleotidase in the tissue. Endogenous norepinephrine (NE) activates both alpha(1)-adrenoceptors and protein kinase C (PKC), which, in turn, activates ecto-5'-nucleotidase via phosphorylation thereby enhancing the production of interstitial adenosine. Histamine-release NE activates PKC, which increased ecto-5'-nucleotidase activity and augmented release of adenosine. Opening of cardiac ATP sensitive K(+) (K(ATP)) channels may cause hydroxyl radical (.OH) generation through NE release. Lysophosphatidylcholine (LPC), an endogenous amphiphiphilic lipid metabolite, also increases the concentration of interstitial adenosine in rat hearts, through the PKC-mediated activation of endogenous ecto-5'-nucleotidase. Nitric oxide (NO) facilitates the production of interstitial adenosine, via guanosine 3',5'-cyclic monophosphate (cGMP)-mediated activation of ecto-5'-nucleotidase as another pathway. These mechanisms play an important role in high sensitivity of the cardiac adenosine system. Adenosine plays an important role as a modulator of ischemic reperfusion injury, and that the production and mechanism of action of adenosine are linked with NE release.     

Adenosine kinase and 5'-nucleotidase activity after prolonged wakefulness in the cortex and the basal forebrain of rat

The effect of prolonged wakefulness on adenosine kinase (AK), ecto-5'-nucleotidase and endo-5'-nucleotidase activity was assessed in the present study. Rats were sleep deprived for 3 or 6h, and one group was allowed to sleep 2h of recovery sleep after the 6h deprivation. The cortex and the basal forebrain were dissected, and frozen rapidly on dry ice. The enzyme activity of adenosine kinase was measured by monitoring the conversion of [2-3H]-adenosine into [3H]-adenosine monophosphate (AMP) and the ecto-5'-nucleotidase and endo-5'-nucleotidase activities by monitoring the conversion of [2-3H]-AMP into [3H]-adenosine. The enzyme activities did not change during deprivation or recovery sleep in either cortex or basal forebrain when compared to unhandled controls. Significant diurnal variation in enzyme activities was noted in both brain areas. In the basal forebrain adenosine kinase and both nucleotidases showed their lowest activity in the middle of the rest phase, 6h after lights on, suggesting a low level of adenosine metabolism, both production and degradation at this time point. In the cortex adenosine kinase had a diurnal activity pattern similar to the basal forebrain and the ecto-5'-nucleotidase activity was low already early in the rest phase, 3h after lights on, and remained low until the end part of the rest phase, 8h after lights on. Endo-5'-nucleotidase lacked diurnal variation. These activity patterns may be associated with the lower level of energy metabolism during sleep compared to wakefulness.     

Cartilage cyclic nucleotide phosphodiesterase: inhibition by anti-inflammatory agents

Soluble 3′,5′-nucleotide phosphodiesterase (PDE) activity is described in chicken epiphyseal and articular cartilage. Kinetic studies of these enzymes demonstrate a high and low K_m for the substrates, adenosine 3′,5′-cyclic monophosphate (cyclic AMP) and guanosine 3′,5′-cyclic monophosphate (cyclic GMP). Epiphyseal and articular PDE activities are inhibited by those anti-inflammatory agents which are potent inhibitors of the enzyme, prostaglandin synthetase (PS). Specificity of this inhibition is indicated by the activity of these agents against the low K_m enzyme. Other anti-inflammatory agents with significantly less potency as PS inhibitors or with no activity against prostaglandin synthetase are found to be either inactive or relatively less potent as inhibitors of cartilage PDE activity. A variety of other anti-inflammatory or anti-rheumatic agents, which are not known to affect prostaglandin synthetase activity, are poor inhibitors of cartilage PDE activity. These data provide insight into the mechanism of action of certain anti-inflammatory agents and into the relationships between prostaglandins and inflammatory reactions.     

Mechanism of cyclic AMP effect on nutrient transport in Chinese hamster ovary cells. A genetic approach

The effect of 8-bromo cyclic adenosine 3':5'-monophosphate (8-Br-cAMP) on sugar and amino acid transport was investigated in wild-type Chinese hamster ovary (CHO) cells and in mutants selected for resistance to cAMP inhibition of cell growth. In wild type cells, both 3-O-methyl-D-glucose and alpha-aminoisobutyric acid transport were decreased in cells treated for 24 h with 8-Br-cAMP; kinetic analysis indicated that a decrease in Vmax, without a significant change in Km, accounted for the lower transport capacity of 8-Br-cAMP treated cells. Among the different transport systems contributing to amino acid entry, "alanine" preferring transport system (system A) appeared to be specifically affected. The sensitivity of transport processes to 8-Br-cAMP was tested in three cAMP-resistant cell lines. When tested for their capacity to phosphorylate histones in crude extracts, one strain had apparently normal amounts of protein kinase activity, one strain had a decreased enzyme sensitivity to cAMP, and one strain had little or no enzyme activity. In all three mutants, no effect of 8-Br-cAMP on 3-O-methyl glucose and alpha-aminoisobutyric acid transport could be observed, regardless of the level of cAMP-dependent protein kinase activity. These data do not indicate whether the effect of cAMP on nutrient transport in CHO cells is the cause or consequence of growth inhibition. However, they support the conclusion that, in CHO cells, the presence of a normally functioning cAMP-dependent protein kinase appears to be necessary but may not be sufficient to observe the effects of cAMP on nutrient transport as well as cell shape and cell growth.     

Effect of medroxyprogesterone acetate on the intestinal absorptive functions in protein-deficient rat

Effect of Medroxyprogesterone acetate (MPA) at a dose level of 35mg/Kg body weight per week for four weeks on the intestinal uptake of nutrients viz glucose, amino acids, (alanine and leucine), calcium and zinc has been investigated in protein-deficient female rats. The administration of MPA was found to enhance significantly the uptake of glucose and amino acids in both the pair-fed and the protein-deficient rats. In contrast, calcium uptake was depressed as a result of treatment with the drug as well as protein-deficiency. The uptake of zinc was not affected on drug administration. This steroidal contraceptive caused elevation in sodium-dependent glucose uptake, while the sodium-independent uptake remained unaltered. The kinetic parameters of glucose and leucine uptake indicate that MPA might be inducing the transport carrier protein of these nutrients as elevation in Vmax of these nutrients transport system was observed following its administration.     

The effect of papain upon proline and sodium transport of rat renal brush-border membrane vesicles

Treatment of renal brush-border membrane vesicles with papain resulted in the removal of the activity of maltase, gamma-glutamyl transpeptidase and leucine aminopeptidase by 85, 50 and 75%, respectively. Stripping of these membrane enzyme activities constituted about 2% of the total membrane proteins and resulted in a widespread diminution in the ability of a variety of amino acids and sugars to be taken up by the membrane vesicles which remained osmotically responsive. Kinetic analysis of the uptake of proline, which was shown previously to be transported by both sodium-dependent and sodium-independent systems, revealed that the Vmax for the sodium-dependent system and Km for the sodium-independent system were halved, but other parameters were not affected indicating that the papain treatment altered sodium-gradient-stimulated entry and the affinity of the sodium-gradient-independent system for proline. Experiments on sodium entry and efflux demonstrate a marked enhancement of flux, so that equilibration of the sodium gradient occurred about 5-times more rapidly than in untreated vesicles. This occurred without any change in the osmotic properties of the vesicle with regard to sodium or amino acid uptake. Studies of fluorescence polarization suggest that incubation with papain does not alter the lipid domains of the membrane.