Influence of pH on the uptake of 5-fluorouracil into isolated tumour cells

To investigate the possible dependence of 5-fluorouracil (5FU) uptake in tumours on the intra- (pHi) and extracellular (pHe) pH, a pH gradient (deltapH) was imposed across the plasma membrane of ascites tumour cells in vitro, similar to that known to occur in some solid tumours in vivo, by incubation in media of PHe 5-8. A > or = 2:1 (intracellular/extracellular) accumulation of radiolabelled 5FU occurred after 5 min incubation of the cells with 0.5 mM 5FU at pHe of 5.0, 5.5 or 6.0. 5FU metabolism is slow under these conditions, and 5FU uptake was not affected by longer incubations up to 20 min, nor by the absence of a sodium gradient. pHi was estimated from the distribution of the weak acid, 5.5-dimethyl-2,4-oxazolidione ([14C]DMO) across the cell membrane. There was significant correlation between the intracellular/extracellular 5FU ratio and pHe (from pHe 6-8), deltapH and pHi (P < 0.02). Similar results were obtained with HT29 cells. Incubation with a drug that made plasma membranes permeable to H+ significantly decreased 5FU uptake in Lettre cells. The co-transport of 5FU may occur on a proton symport using the proton motive force of the deltapH.     

Identification of members of the HSP30 small heat shock protein family and characterization of their developmental regulation in heat-shocked Xenopus laevis embryos

Uptakes of (28)Mg at 10 s were measured at 0.1 and 1mM MgCl(2), to mainly represent one or other of the two uptake mechanisms earlier shown to be present in rat jejunal brush border membrane vesicles, one with an apparent KT of 0.2 mM, the other in the millimolar range. Both mechanisms had an optimal temperature close to 28 degrees C, inactivation at 37 degrees C being more acute for the low affinity mechanism (55 percent, P < 0.01). Both mechanisms were equally stimulated by an electrical potential difference (negative inside the vesicles) imposed by a potassium gradient and not affected by the nature of the anion accompanying magnesium. At 0.1 mM MgCl(2), the uptake was increased by an outwardly directed proton gradient, pH 8.2 outside and 7.4 inside (38 percent, P < 0.05), but not depressed when the gradient was in the opposite direction, pH 6.6 outside and 7.4 inside. It was trans-stimulated by magnesium, strongly inhibited by amiloride and to a smaller extent by furosemide, but uninfluenced by 0.1 mM NaCl or by 100 mM NaCl, NaSCN or KCl. A slight but significant inhibition (20-30 per cent) was recorded in the presence of 0.1 mM CoCl(2), NlCl(2) or BaCl(2). At 1 mM MgCl(2), the uptake was not influenced by pH gradient, was not trans-stimulated by Mg and was not affected by furosemide. A 40 percent inhibition by amiloride was, however, recorded. Also 100 mM NaCl or KCl doubled the uptake, while 1 mM NaCl or 100 mM NaSCN did not affect it. In contrast, all the divalent cations tested produced an inhibition (from 60 to 12 percent) in the following order: Co > or = Mn > Ca > or = Ni> Ba > Sr, when used at the same concentration as magnesium. The results showed that cobalt and calcium were not true competitors. In conclusion, two distinct mechanisms would operate magnesium entry at the brush border: (1) an electrogenic high affinity Mg/Mg,H exchange, sensitive to amiloride and furosemide, and (2) an electrogenic low affinity mechanism, inhibited by the presence of several divalent cations and dependent on the presence or activity of alkaline phosphatase.     

Nicotinic acid transport mediated by pH-dependent anion antiporter and proton cotransporter in rabbit intestinal brush-border membrane

In order to determine whether the vitamin nicotinic acid is absorbed via an anion antiporter, intestinal epithelial cell membrane transport mechanisms for nicotinic acid were characterized using isolated rabbit jejunal brush-border membrane vesicles. The uptake of nicotinic acid by the membrane vesicles showed an overshoot phenomenon in the presence of an outwardly directed bicarbonate gradient or an inwardly directed proton gradient and the uptakes were two times and six times greater, respectively, than that in the absence of any ion gradient. The bicarbonate-dependent initial uptake of nicotinic acid was increased at acidic pH, showing pH-dependent transport activity. An inhibitor of anion transport, 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid, specifically reduced bicarbonate-dependent transport of nicotinic acid. The initial uptakes of nicotinic acid via the anion antiporter and the proton cotransporter were specifically inhibited by monocarboxylic acids such as acetic acid, benzoic acid, D- and L-lactic acid, pravastatin and valproic acid, but not by di- or tricarboxylic acids, bile acids or amino acids. Nicotinic acid uptake activity was, furthermore, expressed in a Xenopus laevis oocyte system after injection of messenger RNA (mRNA) derived from rabbit intestinal epithelial cells. These observations demonstrate that nicotinic acid is absorbed by two independent active transport mechanisms from small intestine, i.e. a proton cotransporter and an anion antiporter. The pH-dependence observed in the intestinal absorption of nicotinic acid might, therefore, be ascribed partly to pH-sensitive and partly to carrier-mediated transport mechanisms in the brush-border membrane.     

Stoichiometry and kinetics of the high-affinity H+-coupled peptide transporter PepT2

Proton-coupled peptide transporters mediate the absorption of a large variety of di- and tripeptides as well as peptide-like pharmacologically active compounds. We report a kinetic analysis of the rat kidney high-affinity peptide transporter PepT2 expressed in Xenopus oocytes. By use of simultaneous radioactive uptake and current measurements under voltage-clamp condition, the charge to substrate uptake ratio was found to be close to 2 for both D-Phe-L-Ala and D-Phe-L-Glu, indicating that the H+:substrate stoichiometry is 2:1 and 3:1 for neutral and anionic dipeptides, respectively. The higher stoichiometry for anionic peptides suggests that they are transported in the protonated form. For D-Phe-L-Lys, the charge:uptake ratio averaged 2.4 from pooled experiments, suggesting that Phe-Lys crosses the membrane via PepT2 either in its deprotonated (neutral) or its positively charged form, averaging a H+:Phe-Lys stoichiometry of 1.4:1. These findings led to the overall conclusion that PepT2 couples transport of one peptide molecule to two H+. This is in contrast to the low-affinity transporter PepT1 that couples transport of one peptide to one H+. Quinapril inhibited PepT2-mediated currents in presence or in absence of external substrates. Oocytes expressing PepT2 exhibited quinapril-sensitive outward currents. In the absence of external substrate, a quinapril-sensitive proton inward current (proton leak) was also observed which, together with the observed pH-dependent PepT2-specific presteady-state currents (Ipss), indicates that at least one H+ binds to the transporter prior to substrate. PepT2 exhibited Ipss in response to hyperpolarization at pH 6.5-8.0. However, contrary to previous observations on various transporters, 1) no significant currents were observed corresponding to voltage jumps returning from hyperpolarization, and 2) at reduced extracellular pH, no significant Ipss were observed in either direction. Together with observed lower substrate affinities and decreased PepT2-mediated currents at hyperpolarized Vm, our data are consistent with the concept that hyperpolarization exerts inactivation effects on the transporter which are enhanced by low pH. Our studies revealed distinct properties of PepT2, compared with PepT1 and other ion-coupled transporters.     

Protein-disulfide isomerase-mediated reduction of the A subunit of cholera toxin in a human intestinal cell line

A key step in the action of cholera toxin (CT) is the reduction of its A subunit to the A1 peptide. The latter is an ADP-ribosyltransferase, which activates the alpha-subunit of the stimulatory G protein of adenylyl cyclase. In this study, the enzymatic reduction of membrane-bound CT in CaCo-2 human intestinal epithelial cells was characterized. Whereas diphtheria toxin was found to be reduced by a cell surface population of protein-disulfide isomerase (PDI) and its cytotoxicity was inhibited by p-chloromercuribenzenesulfonic acid, bacitracin, or anti-PDI antibodies, these inhibitors had no effect on CT reduction or activity in intact cells. In contrast, the reduction of CT in vitro by either postnuclear supernatants (PNS) or microsomal membranes in the presence of Triton X-100 was significantly inhibited by p-chloromercuribenzenesulfonic acid and bacitracin. Anti-PDI monoclonal antibodies likewise inhibited the in vitro reduction of CT and also were effective in depleting reductase activity from PNS. Since inhibition and depletion were not observed in the absence of detergent, these results suggested that the reductase activity was a soluble component localized to the lumen of microsomal vesicles and correlated with the presence of protein-disulfide isomerase. This was further confirmed by showing a corresponding depletion of reductase activity and PDI in alkali-treated microsomes. This activity was restored when purified bovine PDI was added back to alkali-treated microsomes in a redox buffer that reflected conditions found in the lumen of the endoplasmic reticulum (ER). When the CT-related reductase activity was assayed in subcellular fractions of PNS-derived membranes isolated on a 9-30% Iodixanol gradient, the activity, as measured by CT-A1 peptide formation localized to those fractions containing PDI. Likewise CT-A1 peptide formed in intact cells co-localized to those membrane fractions containing the majority of cellular PDI. Furthermore, the banding density corresponded to a region of the gradient containing ER-derived membranes. These results indicated that CT was a substrate for PDI-catalyzed reduction in intact cells and supported the hypothesis that CT reduction and activation occurs in the ER.     

Proton-cotransport of pravastatin across intestinal brush-border membrane

PURPOSE: The purpose of the present study is to clarify the intestinal brush-border transport mechanism of a weak organic acid, pravastatin, an HMG-CoA reductase inhibitor. METHODS: The transport of pravastatin was studied by using intestinal brush-border membrane vesicles prepared from rabbit jejunum, and uptake by the membrane vesicles was measured using rapid filtration technique. RESULTS: The initial uptake of [14C]pravastatin was markedly increased with decreases in extravesicular pH and showed a clear overshoot phenomenon in the presence of a proton gradient (pHin/out = 7.5/5.5). A protonophore, carbonylcyanide p-trifluoromethoxyphenylhydrazone, significantly reduced the uptake of [14C]pravastatin. In addition, an ionophore for sodium, potassium and proton, nigericin, stimulated the uptake of [14C]pravastatin in the presence of a potassium gradient ([K+]in/[K+]out = 0/145 mM). On the other hand, neither the imposition of an inwardly directed sodium gradient nor an outwardly directed bicarbonate gradient stimulated the uptake of [14C]pravastatin. In the presence of a proton gradient (pHin/out = 7.5/5.5), the initial uptake of pravastatin was saturable with the apparent Kt of 15.2 +/- 3.2 mM and Jmax of 10.6 +/- 1.21 nmol/mg protein/10 sec. The uptake of pravastatin was significantly inhibited by monocarboxylic acid compounds such as acetic acid and nicotinic acid in a competitive manner but not by di- or tricarboxylic acids, or acidic amino acid. CONCLUSION: It was concluded that a pH-dependent transport of pravastatin across the brush-border membrane occurs by a proton-gradient dependent carrier-mediated mechanism rather than by simple diffusion of its unionized form.     

Changes in transforming growth factor beta1 gene expression and immunoreactivity levels during development of chronic radiation enteropathy

Previously, we reported that pirarubicin (THP), an anthracycline, was taken up, at least in part, by both human leukemic HL60 cells and mononuclear cells (MNCs) via a carrier-mediated system. In this study, the possibility of a contribution of nucleoside transport systems to the uptake of THP by HL60 cells and MNCs was investigated. The experiments were performed after both types of cells had been pretreated with a metabolic inhibitor, 2,4-dinitrophenol, to deplete cellular ATP. In HL60 cells, THP uptake was increased and decreased significantly by treatment with equilibrative nucleoside transport inhibitors, nitrobenzylthioinosine (NBMPR), nitrobenzylthioguanosine and dilazep, in the presence and absence, respectively, of an inwardly directed Na+-gradient. THP uptake by HL60 cells showed an overshoot in the presence of the gradient, and was decreased by treatment of the cells with monensin, indicating that the uptake partially depended on the Na+-gradient. In HL60 cells in which equilibrative nucleoside transport was inhibited by NBMPR, THP uptake in the presence of the gradient was inhibited by Na+-dependent concentrative nucleoside transport inhibitors, but no inhibition was observed in the absence of the gradient. In MNCs, conversely, there was no effect of any equilibrative nucleoside transport inhibitor or the Na+-gradient on THP uptake. These results suggested that THP was taken up, at least in part, via both equilibrative and concentrative nucleoside transport systems in HL60 cells, but not in MNCs.     

Regional distribution of vascular resistance in two models of experimental renovascular hypertension

The data from 88 patients (pts) with aortic stenosis (AS) were reviewed to determine relationships between angina pectoris (AP) and coronary artery disease (CAD). Results of surgery performed in 81 of these pts was analyzed. All pts had coronary arteriograms, and lesions greater than or equal to 50% were considered significant. Fifty-nine pts had an aortic valve gradient measured at catheterization greater than or equal to 40 mmHg, and in 29 pts, AS was confirmed at operation. Sixty-eight pts (77%) experienced AP, and 32 had coexisting CAD (47%); 9 of 20 pts without AP had CAD (45%). There were no significant differences in the incidence of AP in pts divided into subgroups by the aortic valve gradient (40-50, 51-100, 101-200 mmHg) or age (40-59, 60-81 years). Also, no significant differences were found in the incidence or extent of CAD between the two age groups; the extent of CAD was similar regardless of the presence or absence of AP. In pts with AP (1) CAD was more likely in pts greater than or equal to 60 years of age; (2) CAD was less likely when the aortic valve gradient was greater than 100 mmHg, suggesting that AP in these pts was due to hemodynamically severe AS. All pts with 3-vessel CAD experienced AP, and the aortic valve gradient was less in these pts than in those with no CAD or less extensive CAD. In 19 pts with combined AS and CAD who had both the aortic valve replaced and a revascularization operation only 1 of pts died in the hospital, while 3 of 19 pts with combined AS and CAD who had aortic valve replacement alone died. In this study a significant number of pts with AS experienced AP, and the presence or absence of AP did not predict coexisting CAD. Coronary arteriography is recommended in the evaluation of pts greater than or equal to 40 years of age with AS. The operative mortality appears to be decreased in pts with AS and CAD who have combined surgery.     

Determinants of transmission success of individual clones from mixed-clone infections of the rodent malaria parasite, Plasmodium chabaudi

PURPOSE: To compare the permeation characteristics of amide bond-containing HIV-1 protease inhibitors and their pyrrolinone-containing counterparts across Caco-2 cell monolayers, a model of the intestinal mucosa. METHODS: Transepithelial transport and cellular uptake of three pairs of amide bond-containing and pyrrolinone-based peptidomimetics were assessed in the presence and absence of cyclosporin A using the Caco-2 cell culture model. The potential of the peptidomimetics to interact with biological membranes was estimated by IAM chromatography. RESULTS: In the absence of cyclosporin A, apical (AP) to basolateral (BL) flux of all compounds studied was less than the flux determined in the opposite direction (i.e., BL-to-AP). The ratio of the apparent permeability coefficients (Papp) calculated for the BL-to-AP and AP-to-BL transport (P(BL-->AP)/P(AP-->BL)) varied between 1.7 and 36.2. When individual pairs were ompared, P(BL-->AP)/P(AP-BL) ratios of the pyrrolinone-containing compounds were 1.5 to 11.5 times greater than those determined for the amide bond-containing analogs. Addition of 25 microM cyclosporin A to the transport buffer reduced the P(BL-->AP)/P(AP-->BL) ratios for all protease inhibitors to a value close to unity. Under these conditions, the amide bond-containing peptidomimetics were at least 1.6 to 2.8 times more able to permeate Caco-2 cell monolayers than were the pyrrolinone-containing compounds. The intrinsic uptake characteristics into Caco-2 cells determined in the presence of 25 microM cyclosporin A were slightly greater for the amide bond-containing protease inhibitors than for the pyrrolinone-containing analogs. These uptake results are consistent with the transepithelial transport results determined across this in vitro model of the intestinal mucosa. CONCLUSIONS: The amide bond-containing and pyrrolinone-based peptidomimetics are substrates for apically polarized efflux systems present in Caco-2 cell monolayers. The intrinsic permeabilities of the amide bond-containing protease inhibitors are slightly greater than the intrinsic permeabilities of the pyrrolinone-based analogs through Caco-2 cell monolayers.     

Dipeptide uptake and transport characteristics in rabbit tracheal epithelial cell layers cultured at an air interface

PURPOSE: To determine the functional presence ofa H+/peptide cotransport process in rabbit tracheal epithelial cell layers cultured at an air-interface and its contribution to transepithelial dipeptide transport. METHODS: Rabbit tracheocytes were isolated, plated on Transwells, and cultured at an air-interface. After 5 or 6 days in culture, uptake and transepithelial transport of carnosine were examined. RESULTS: Carnosine uptake by tracheocytes was pH-dependent and was saturable with a Michaelis-Menten constant of 170 microM. Moreover, carnosine uptake was inhibited 94% by Gly-L-Phe, 28% by beta-Ala-Gly, but not at all by Gly-D-Phe or by the amino acids beta-Ala and L-His. Unexpectedly. transepithelial carnosine transport at pH 7.4 (i.e., in the absence of a transepithelial pH gradient) was similar in both the apical-to-basolateral (ab) and basolateral-to-apical (ba) directions. Lowering the apical fluid pH to 6.5 reduced ab transport 1.6 times without affecting ba transport, consistent with predominantly paracellular diffusion of carnosine under an electrochemical potential gradient. CONCLUSIONS: The kinetic behavior of carnosine uptake into cultured tracheal epithelial cell layers is characteristic of a H+-coupled dipeptide transport process known to exist in the small intestine and the kidney. Such a process does not appear to be rate-limiting in the transport of carnosine across the tracheal epithelial barrier.     

Nuclear binding of estradiol in human myometrium

This study confirmed the changed binding capacity in human myometrium of receptors in the presence and absence of diisopropylfluorophosphate (DFP) and investigated the nuclear uptake of the different receptor forms present in cytosol fractions. A low-salt medium was used. The radiolabled (hydrogen) estradiol receptor complex in the cytosol prepared in the presence of DFP had much greater nuclear binding activity than the same complexes prepared in the absence of DFP. The difference in nuclear translocation was obtained despite greater binding capacity of receptors in cytosol prepared in absence of DFP. Receptors prepared with DFP sedimented at 9S, 5.3S, and 4.2S. Sucrose density gradient centrifugation of cytoplasmic receptors after incubation with nuclei showed that the radioactivity sedimenting at 9S disappeared but the other 2 sedimentation complexes were unchanged. Nuclear uptake of radiolabled estradiol receptor complex from cytosol prepared in the absence of DFP was also dependent on the presence of the 9S complex, whereas the 4.2S proteolytic fragment did not show any nuclear binding. A correlation coefficient of .99 was obtained when the decrease in amount of 9S receptor form was plotted against the amount of radiolabled estradiol extracted from the nuclear pellet.     

A reexamination of the role of imagery in learning and memory.

Five experiments were conducted to examine whether the superior recall of concrete over abstract words might be better accounted for in terms of relative differences in the processing of relational and distinctive information rather than redundant verbal and imaginal memory codes. Concrete and abstract word pairs were presented in the standard paired-associated learning task or under conditions intended to affect the nature and extent of relational processing between pair members. Concreteness effects were attenuated or eliminated when relational processing was prevented at encoding (Experiments 3, 4, and 5) or when the use of encoded relations within pairs was prevented at recall (Experiments 1, 2, and 3). The results indicated the viability of an account of concreteness effects in paired-associate learning based on the joint functions of distinctive and relational information. They also remove theoretical constraints imposed on imagery theories by the incorrect assumption of a uniform presence of concreteness effects in memory for word lists. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Inhibitory effects of angiotensin-converting enzyme inhibitor on cefroxadine uptake by rabbit small intestinal brush border membrane vesicles

Effects of angiotensin-converting enzyme (ACE) inhibitors, captopril, enalapril maleate and quinapril, on the uptake of aminocephalosporin antibiotic, cefroxadine, by rabbit small intestinal brush border membrane vesicles were examined. These ACE inhibitors significantly inhibited the uptake of cefroxadine, which is transported by H+/dipeptide transporter in the membrane, in the order of captopril < enalapril < quinapril in the presence of an inward H+ gradient. Inhibitory effect of quinapril was more marked than that of aminocephalosporin cephradine, while in the absence of an inward H+ gradient inhibition by these ACE inhibitors was much less. Dixon plot analysis showed that the inhibition by enalapril and quinapril in the presence of an inward H+ gradient occurred in a competitive manner and estimated inhibition constants of these two drugs to be 5.3 mM and 0.46 mM, respectively. These results suggested the strong affinity of these ACE inhibitors, especially quinapril, on the H+/dipeptide transporter.     

Ethical issues in stem cell transplantation

Citrate transport into the vacuoles of acid lime juice cells was investigated using isolated tonoplast vesicles. ATP stimulated citrate uptake in the presence or in the absence of a Delta mu H+. Energization of the vesicles only by an artificial K+ gradient (establishing an inside-positive Delta psi) also resulted in citrate uptake as was the case of a Delta pH dominated Delta mu H+. Addition of inhibitors to endomembrane ATPases showed no direct correlation between the inhibition to the tonoplast bound H+/ATPase and citrate uptake. The data indicated that, although some citrate uptake can be accounted for by Delta psi and by a direct primary active transport mechanism involving ATP, under in vivo conditions of vacuolar pH of 2.0, citrate uptake is driven by Delta pH.     

Replacement of troponin-I in slow-twitch skeletal muscle alters the effects of the calcium sensitizer EMD 53998

A component of fungus Thielavia minor, OPC-15161, has been shown to inhibit the proliferation and extracellular matrix production of extracellular matrix-producing mesangial cells in the kidney in vivo. In this study, we examined the effects of OPC-15161 on the proliferation and extracellular matrix production of rat cultured hepatic stellate cells (HSCs). To determine the effect of OPC-15161 on proliferation of HSCs, the cell number and the uptake of [3H]thymidine were investigated in the presence and absence of interleukin-1beta (IL-1beta). IL-1beta significantly increased the uptake of [3H]thymidine in the HSCs, and the addition of OPC-15161 inhibited the uptake in a dose-dependent manner. The cell number of HSCs was also increased by IL-1beta, which was inhibited by OPC-15161. Production of extracellular matrix by OPC-15161 was studied by the production of [3H]-hydroxyproline in the presence and absence of transforming growth factor-beta1 (TGF-beta1). TGF-beta1 significantly increased the production of [3H]-hydroxyproline in the cells, whereas the addition of OPC-15161 inhibited this effect dose dependently. We also investigated the effects of OPC-15161 on Ca2+ mobilization and measured D-myo-inositol 1,4,5-triphosphate (IP3) in the HSCs. IL-1beta induced the increase of intracellular Ca2+ and IP3 concentrations in the HSCs, which were decreased by OPC-15161. Based on these results, we conclude that OPC-1 5161 inhibited the proliferation and production of hydroxyproline in cultured rat HSCs, and thus, it may have a role in prevention of liver fibrosis in vivo.     

The efflux of lysine from the basolateral membrane of human cultured intestinal cells (Caco-2) occurs by different mechanisms depending on the extracellular availability of amino acids

The efflux of the nutritionally essential amino acid, L-lysine from the basolateral (BL) membrane was characterized in human cultured intestinal cells (Caco-2) grown and differentiated on permeable filter supports. Cells were loaded by incubating with 3H-lysine from the apical (AP) side in the absence of sodium (substituted with choline) in the BL medium; under these conditions, cells accumulated lysine in the intracellular soluble pool to 10- to 20-fold the extracellular concentration. L-Lysine efflux in the BL medium was then followed, and initial rates of efflux were calculated under different experimental conditions. L-Lysine efflux exhibited a strong energy dependence. The presence of an inwardly directed gradient of sodium or lithium stimulated lysine efflux; ouabain reduced efflux in both sodium- and lithium-containing medium. When zwitterionic or cationic amino acids were added to the BL medium, L-lysine efflux was strongly stimulated. The most efficient trans-stimulating amino acids were L-leucine > L-methionine = L-ornithine = L-arginine. In the presence of trans-stimulating amino acids in the BL medium, L-lysine efflux exhibited energy independence and was not affected by the presence of a sodium gradient. In addition, the sensitivity, of efflux to N-ethylmaleimide was different in the absence or in the presence of amino acids in the BL medium. These results suggest that different mechanisms may operate in the BL efflux of L-lysine from human intestinal epithelial cells, depending on the extracellular availability of other amino acids, to guarantee optimal bioavailability of this essential amino acid both in the postprandial absorptive period and between meals.     

Sodium ion-substrate symport in a marine bacterium

Anaerobic suspensions of Alteromonas haloplanktis accumulated alpha-aminoisobutyric acid, by a sodium-dependent process, in response to an artificially imposed membrane potential in the presence or absence of a transmembrane chemical gradient of sodium. These results suggest that the transport of alpha-aminoisobutyrate by this organism occurs via Na+-substrate symport.     

Effect of glucagon on pinocytosis by the yolk sac of the rat

The uptake of macromolecular markers by fluid pinocytosis in the rat yolk sac was inhibited by glucagon, with half-maximal effect at a hormone concentration of approximately 3 X 10(-8) M. Glucagon had no effect on the cellular distribution of the marker subsequent to its uptake. Rates of uptake promptly returned to normal when the yolk sacs were transferred from a glucagon-containing to a glucagon-free medium. Epinephrine also inhibited, but only at much higher concentrations. The effect of the latter was augmented by theophylline. Insulin (10(-6) M) had no effect when added alone or with an inhibitory level of glucagon (10(-7) M). The presumption that the hormone effect was mediated by cyclic AMP was supported by the findings that the cellular levels of cyclic AMP were elevated in the presence of glucagon and that dibutyryl cyclic AMP could replace glucagon as an effective inhibitor. The conclusion that the hormone effect was on uptake rather than on subsequent regurgitation was based on the linearity of accumulation in both the presence and absence of glucagon and the inability of glucagon to stimulate loss of invertase from preloaded cells. Colchicine and vinblastine also inhibited uptake. This finding and those of others which are discussed suggest the possibility that effects of cyclic nucleotides on certain cell functions may involve their regulation of microtubular status.     

Detection of subtle abnormalities on chest radiographs after irreversible compression

In both oxidative phosphorylation and photo-phosphorylation, electron flow through a carrier is linked to the generation of ATP. The energy released by electron transport is converted to potential energy forming a proton gradient across the membranes in chloroplasts. The proton gradient can be measured by a pH microelectrode. In this report, pH changes produced by photo-induced proton transport through spinach chloroplast membranes were measured by a glass microelectrode. The effect of 5,5'-dithiobis(2-nitrobenzoate) (DTNB) on the kinetics of proton movement across the thylakoid membranes was studied. The results showed that the rate of proton uptake was reduced with increasing DTNB concentration. The rate of leakage of accumulated protons through thylakoid membranes also decreased. The results support the notion that cysteinyl residue is involved in proton translocation. The inhibition of proton transport would subsequently affect the chemical reactions of the Calvin Cycle that takes place in the stroma which is the soluble compartment surrounding the thylakoid membranes.     

Inhibitory action of extracellular adenosine 5'-triphosphate on parietal cells isolated from rabbit gastric mucosa

The effects of the purine nucleotides, adenosine 5'-triphosphate (ATP) and their analogs 2-methylthio ATP and beta, gamma-methylene ATP, as well as those of the pyrimidine nucleotide, uridine 5'-triphosphate (UTP), on acid production in isolated rabbit gastric parietal cells prepared by enzymatic dispersion and enriched by counterflow elutriation were studied. The (14C)-aminopyrine (AP) accumulation method was used as an index of acid production by the parietal cells. In histamine-stimulated parietal cells, ATP and 2-methylthio ATP, but not beta, gamma-methylene ATP or UTP, produced significant and concentration-related inhibition of the histamine-stimulated AP uptake. The rank order of potency of these nucleotides in inhibiting histamine-stimulated AP accumulation was 2-methylthio ATP > ATP > > beta, gamma-methylene ATP, UTP. In contrast to these results, the AP accumulation responses to secretagogues other than histamine such as carbachol and dibutyryl-cAMP, were not significantly modified by ATP and analogs. Pretreatment of parietal cells with indomethacin, a prostaglandin synthesis inhibitor, led to a significant reduction of the inhibitory responses elicited by ATP on histamine-stimulated AP uptake. These data suggest that ATP selectively inhibits the histamine-stimulated gastric acid secretion in rabbits by acting directly on parietal cells; that a component of this action seems to be related with a stimulation of prostaglandin production; and that the antisecretory effect of ATP on isolated rabbit parietal cells may be mediated via P2Y-purinoceptors.