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Biomedical subjects

S L Bonting

Publications and source records attributed to S L Bonting.

At least 19 recordsLinked to original sources
PubMed1992

Animal research facility for Space Station Freedom.

An integrated animal research facility is planned by NASA for Space Station Freedom which will permit long-term, man-tended experiments on the effects of space conditions on vertebrates. The key element in this facility is a standard type animal habitat which supports and maintains the animals under full bioisolation during transport and during the experiment. A holding unit accommodates the habitats with animals to be maintained at zero gravity; and a centrifuge, those to be maintained at artificial gravity for control purposes or for gravity threshold studies. A glovebox permits handling of the animals for experimental purposes and for transfer to a clean habitat. These facilities are described, and the aspects of environmental control, monitoring, and bioisolation are discussed.

Animals↗
PubMed1992

Utilization of biosensors and chemical sensors for space applications.

There will be a need for a wide array of chemical sensors for biomedical experimentation and for the monitoring of water and air recycling processes on Space Station Freedom. The infrequent logistics flights of the Space Shuttle will necessitate onboard analysis. The advantages of biosensors and chemical sensors over conventional analysis onboard spacecraft are manifold. They require less crew time, space, and power. Sample treatment is not needed. Real time or near-real time monitoring is possible, in some cases on a continuous basis. Sensor signals in digitized form can be transmitted to the ground. Types and requirements for chemical sensors to be used in biomedical experimentation and monitoring of water recycling during long-term space missions are discussed.

Aerospace Medicine↗
PubMed1992

Chemical sensors for space applications.

There will be a great need for a wide variety of chemical analyses, both for biomedical experimentation and for the monitoring of water and air recycling processes on Space Station Freedom and later long-term space missions. The infrequent logistics flights of the Space Shuttle will necessitate onboard analysis. Chemical sensors offer several advantages over conventional analysis onboard a spacecraft. They require less crew time, space, and power. A chemical sensor consists of a selector which selectively interacts with the analyte present in a mixture of substances, and a transducer which produces an electric signal in response to the interaction of analyte and selector. The transducer signal thus provides a quantitative and selective measurement of the analyte. Types and requirements for chemical sensors to be used in biomedical experimentation and monitoring of water recycling during long-term space missions are discussed. With chemical sensors, a wide variety of analytes can be determined selectively without separation steps. In principle, chemical sensors can provide (near) real-time monitoring of many important analytes. In some cases they can even provide continuous monitoring of such analytes. The sensors, and even the ancillary instruments, are small compared to conventional analytical instruments. Their power consumption is low. Sensor measurements do not require extensive sample treatment before analysis. In most cases a sensor can simply be inserted in, or be attached to, the organism; or be placed in the water flowing through the water recycling system. Since the sensor signal can usually be provided in digitized form, rapid transmission to the ground is possible. The use of sensors thus provides an efficient use of the scarce resources of crew time, pressurized volume, and power.

Biosensing Techniques↗
PubMed1988

Is there a Cl- pump?

Three universally accepted mechanisms of Cl- transport across plasma membranes exist and they are 1) anion-coupled antiport, 2) cation-coupled symport, and 3) coupling to primary active ion transport through electrical and/or chemical processes. No unequivocal direct evidence has been provided for primary active Cl- transport (Cl- pump) despite numerous reports of cellular Cl- -stimulated adenosinetriphosphatase (ATPases) and of Cl- transport that cannot be accounted for by the three well-documented Cl- transport processes. It has been demonstrated that Cl- -stimulated ATPase activity is localized to both mitochondrial and microsomal aspects of the cellular apparatus. However, one group ascribes microsomal localization of Cl- -stimulated ATPase activity to mitochondrial contamination of that membrane fraction. Therefore, no Cl- pump could ever exist naturally in any plasma membrane. The other group simply states that there is plasma membrane localization of Cl- -stimulated ATPase activity that could function as a Cl- pump. Both arguments are logically advanced and their conclusions are consistent with their respective premises. Resolution to the question Is there a Cl- pump? rests with each reader's critique and objective evaluation.

Acetabularia↗
PubMed1986

Sodium and buffer cations inhibit dephosphorylation of (Na+ + K+)-ATPase.

Effects of various cations on the dephosphorylation of (Na+ + K+)-ATPase, phosphorylated by ATP in 50 mM imidazole buffer (pH 7.0) at 22 degrees C without added Na+, have been studied. The dephosphorylation in imidazole buffer without added K+ is extremely sensitive to K+-activation (Km K+ = 1 microM), less sensitive to Mg2+-activation (Km Mg2+ = 0.1 mM) and Na+-activation (Km Na+ = 63 mM). Imidazole and Na+ effectively inhibit K+-activated dephosphorylation in linear competitive fashion (Ki imidazole 7.5 mM, Ki Na+ 4.6 mM). The Ki for Na+ is independent of the imidazole concentration, indicating different and non-interacting inhibitory sites for Na+ and imidazole. Imidazole inhibits Mg2+-activated dephosphorylation just as effective as K+-activated dephosphorylation, as judged from the Ki values for imidazole in the two processes. Tris buffer and choline chloride, like imidazole, inhibit dephosphorylation in the presence of residual K+ (less than 1 microM), but less effectively in terms of I50 values and extent of inhibition. Tris inhibits to the same extent as choline. This indicates different inhibitory sites for Tris or choline and for imidazole. These findings indicate that high steady-state phosphorylation levels in Na+-free imidazole buffer are due to the induction of a phosphorylating enzyme conformation and to the inhibition of (K+ + Mg2+)-stimulated dephosphorylation.

Animals↗
PubMed1986

Free protons do not substitute for sodium ions in buffer-mediated phosphorylation of (Na+ + K+)-ATPase.

In view of our recent finding of imidazole-activation of the phosphorylation of (Na+ + K+)-ATPase and the suggestion by others of an activating role of protons, in lieu of sodium ions, in the overall hydrolytic and phosphorylation processes of the enzyme, we have investigated the effect of pH on the phosphorylation process. No indication of proton activation is found. Rather, phosphorylation at low pH in the absence of Na+ is dependent on the buffer concentration. Imidazole-H+ stimulated phosphorylation at pH 5 reaches the same maximal steady-state level as Na+-stimulated phosphorylation. Low pH also elicits Tris-H+ stimulated phosphorylation, but due to a simultaneous inhibitory effect of this buffer the maximal steady-state level is no more than 50% of the Na+-stimulated phosphorylation level. Protons inhibit rather than activate phosphorylation. Upon decreasing the pH from 7 to 5, we observe for all ligands, whether activating or inhibiting phosphorylation (ATP, Na+, protonated imidazole, Mg2+ and K+), a decrease in affinity (largest for Mg2+) and a decrease in the maximal steady-state phosphorylation capacity. The effects of Na+ and imidazole-H+ on the phosphorylation step have been compared with those on the E2----E1 conformational change, which leads to the phosphorylation step. The different pH-dependence of the affinities for Na+ and protonated buffer in the E2----E1 transition suggests that there are separate activation sites with different pK values for Na+ and the buffer cation. The above findings rule out a role of free protons as a substitution for Na+ in the phosphorylation process.

Adenosine Triphosphate↗
PubMed1985

Direct evidence for an ADP-sensitive phosphointermediate of (K+ + H+)-ATPase.

Direct evidence for the occurrence of an ADP-sensitive phosphoenzyme of (K+ + H+)-ATPase, the proton-pumping system of the gastric parietal cell is presented. The enzyme is phosphorylated with 5 microM [gamma-32P]ATP in 50 mM imidazole-HCl (pH 7.0) and in the presence of 7-15 microM Mg2+. Addition of 5 mM ADP to this preparation greatly accelerates its hydrolysis. We have been able to establish this by stopping the phosphorylation with radioactive ATP, by adding 1 mM non-radioactive ATP, which leads to a slow monoexponential process of dephosphorylation of 32P-labeled enzyme. The relative proportion of the ADP-sensitive phosphoenzyme is 22% of the total phosphoenzyme. Values for the rate constants of breakdown and interconversion of the two phosphoenzyme forms have been determined.

Adenosine Diphosphate↗
PubMed1985

The locus of nucleotide specificity in the reaction mechanism of (Na+ + K+)-ATPase determined with ATP and GTP as substrates.

ATP and GTP have been compared as substrates for (Na+ + K+)-ATPase in Na+-activated hydrolysis, Na+-activated phosphorylation, and the E2K----E1K transition. Without added K+ the optimal Na+-activated hydrolysis rates in imidazole-HCl (pH 7.2) are equal, but are reached at different Na+ concentrations: 80 mM Na+ for GTP, 300 mM Na+ for ATP. The affinities of the substrates for the enzyme are widely different: Km for ATP 0.6 microM, for GTP 147 microM. The Mg-complexed nucleotides antagonize activation as well as inhibition by Na+, depending on the affinity and concentration of the substrate. The optimal 3-s phosphorylation levels in imidazole-HCl (pH 7.0) are equally high for the two substrates (3.6 nmol/mg protein). The Km value for ATP is 0.1-0.2 microM and for GTP it ranges from 50 to 170 microM, depending on the Na+ concentration. The affinity of Na+ for the enzyme in phosphorylation is lower with the lower affinity substrate: Km (Na+) is 1.1 mM with ATP and 3.6 mM with GTP. The GTP-phosphorylated intermediate exists, like the ATP-phosphorylated intermediate, in the E2P conformation. Addition of K+ increases the optimal hydrolytic activity 30-fold for ATP (at 100 mM Na+ + 10 mM K+) and 2-fold for GTP (at 100 mM Na+ + 0.16 mM K+). K+ greatly increases the Km values for both substrates (to 430 microM for ATP and 320 microM for GTP). Above 0.16 mM K+ inhibits GTP hydrolysis. GTP does not reverse the quenching effect of K+ on the fluorescence of the 5-iodoacetamidofluorescein-labeled enzyme. ATP fully reverses this effect, which represents the transition from E1K to E2K. Hence GTP is unable to drive the E2K----E1K transition.

Adenosine Triphosphate↗
PubMed1985

Na+-like effect of imidazole on the phosphorylation of (Na+ + K+)-ATPase.

A high basal level of phosphorylation (approx. 70% of the optimal Na+-dependent phosphorylation level) is observed in 50 mM imidazole-HCl (pH 7.0), in the absence of added Na+ and K+ and the presence of 10-100 microM Mg2+. In 50 mM Tris-HCl (pH 7.0) the basal level is only 5%, irrespective of the Mg2+ concentration. Nevertheless, imidazole is a less effective activator of phosphorylation than Na+ (Km imidazole-H+ 5.9 mM, Km Na+ 2 mM under comparable conditions). Imidazole-activated phosphorylation is strongly pH dependent, being optimal at pH less than or equal to 7 and minimal at pH greater than or equal to 8, while Na+-activated phosphorylation is optimal at pH 7.4. This suggests that imidazole-H+ is the activating species. Imidazole facilitates Na+-stimulated phosphorylation. The Km for Na+ decreases from 0.63 mM at 5 mM imidazole-HCl to 0.21 mM at 50 mM imidazole-HCl (pH 7; 0.1 mM Mg2+ in all cases). Imidazole-activated phosphorylation is more sensitive to inhibition by K+ (I50 = 12.5 microM) than Na+-activated phosphorylation (I50 = 180 microM). Mg2+ antagonizes activation by imidazole-H+ and also inhibition by K+. The Ki value for Mg2+ (approx. 0.3 mM) is the same for the two antagonistic effects. Tris buffer (pH 7.0) inhibits imidazole-activated phosphorylation with an I50 value of 30 mM in 50 mM imidazole-HCl (pH 7.0) plus 0.1 mM Mg2+. We conclude that imidazole-H+, but not Tris-H+, can replace Na+ as an activator of ATP-dependent phosphorylation, primarily by shifting the E2----E1 transition to the right, leading to a phosphorylating E1 conformation which is different from that in Tris buffer.

Animals↗
PubMed1985

Tight junctional permeability of the resting and carbachol stimulated exocrine rabbit pancreas.

The permeability of the pancreatic epithelium to horseradish peroxidase is investigated in the resting and carbachol stimulated rabbit pancreas. Horse radish peroxidase administered to the bathing medium of the isolated rabbit pancreas appears in the secreted fluid of the pancreas in a relatively low concentration. Carbachol stimulates both protein secretion and the passage of horse radish peroxidase into the secretory fluid. Histochemical assessment shows that horseradish peroxidase enters the interstitial spaces of the pancreatic tissue and is present along basal and lateral plasma membranes of acinar and ductular cells. In the absence of carbachol, horseradish peroxidase is seen more frequently in the tight junctions of ductular cells than in those of acinar cells. However, in the carbachol stimulated gland horseradish peroxidase is observed in the junctions between adjacent acinar cells more frequently than in the unstimulated gland. Freeze-fracture of acinar cells shows that the number of tight junctional strands and the tight junction depth are slightly decreased upon carbachol stimulation. The findings suggest that cholinergic stimulation of the exocrine pancreas increases the permeability of the acinar cell junctions to moderately large molecules such as horseradish peroxidase. This may result in an increase of the concentration of the molecule in the secreted fluid.

Animals↗
PubMed1984

The mechanism of fluid secretion in the rabbit pancreas studied by means of various inhibitors.

In order to increase our understanding of the mechanism of pancreatic fluid secretion we have studied the effects of various transport inhibitors on this process in the isolated rabbit pancreas. In this preparation, a high rate of unstimulated fluid secretion occurs, which probably originates from the ductular cells. Inhibitory are ouabain, furosemide, bumetanide, piretanide, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) and acetazolamide, with their half-inhibitory concentrations: 2 X 10(-6) M (ouabain), 1.3 X 10(-3) M (furosemide), 2.2 X 10(-3) M (bumetanide and piretanide) and 1.4 X 10(-4) M (SITS). With acetazolamide a maximal inhibition of only 20% is found at 10(-3) M. Amiloride (10(-3) M) has no effect on pancreatic fluid secretion. The inhibitory effects on HCO-3 output are always larger and those on Cl- output lower than those on fluid secretion. The results suggest that the ouabain-sensitive (Na+ + K+)-ATPase system provides the energy for a Na+-gradient-driven Cl--HCO-3-exchange transport system, sensitive to the loop diuretics furosemide, bumetanide and piretanide and to SITS. This system would drive the transcellular transport of HCO-3 and secondarily that of cations, Cl- and water.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1984

Potentiating role of cyclic AMP in pancreatic enzyme secretion, demonstrated by means of forskolin.

The role of cyclic AMP in the regulation of enzyme secretion by the rabbit pancreas has been investigated by means of forskolin, an activator of the catalytic subunit of adenylate cyclase. Forskolin increases the cyclic AMP level in isolated pancreatic acini in a dose-dependent way. Basal amylase release, however, remains unchanged. Forskolin potentiates the increase in amylase release induced by the C-terminal octapeptide of cholecystokinin (CCK-8). Potentiation is already apparent at hormone concentrations which are only marginally effective in stimulating amylase secretion. CCK-8 alone does not raise the cellular cAMP level, but it potentiates the forskolin-induced increase. In relative terms, potentiation is higher with decreasing concentration of forskolin. These results indicate that cAMP alone does not play a direct role in CCK-stimulated pancreatic enzyme secretion in the rabbit, but it potentiates enzyme secretion already stimulated through a cAMP-independent process.

Amylases↗
PubMed1984

Anion secretion by the isolated rabbit pancreas.

The isolated rabbit pancreas secretes a fluid containing chloride and bicarbonate in about equal concentrations. Replacement of bicarbonate by acetate, phosphate or isethionate, replacement of Na+ by Li+ and addition of ouabain to the bathing medium of the pancreas inhibit the secretion of fluid, chloride and bicarbonate in a similar fashion and by maximally 100%. Replacement of chloride by isethionate inhibits fluid secretion by maximally 50%, chloride secretion by 90% and bicarbonate secretion by 20%. It is concluded that fluid secretion is based on a Na+-gradient-dependent bicarbonate influx or proton efflux in the ductular cell, and that the secretion of chloride is secondary to that of bicarbonate.

Animals↗
PubMed1984

Thiophosphorylation of (Na + K+)-ATPase yields an ADP-sensitive phosphointermediate.

1) Treatment of (Na+ + K+)-ATPase from rabbit kidney outer medulla with the gamma-35S labeled thio-analogue of ATP in the presence of Na+ + Mg2+ and the absence of K+ leads to thiophosphorylation of the enzyme. The Km value for [gamma-S]ATP is 2.2 microM and for Na+ 4.2 mM at 22 degrees C. Thiophosphorylation is a sigmoidal function of the Na+ concentration, yielding a Hill coefficient nH = 2.6. (2) The thio-analogue (Km = 35 microM) can also support overall (Na+ + K+)-ATPase activity, but Vmax at 37 degrees C is only 1.13 mumol X (mg protein)-1 X h-1 or 0.09% of the specific activity for ATP (Km = 0.43 mM). (3) The thiophosphoenzyme intermediate, like the natural phosphoenzyme, is sensitive to hydroxylamine, indicating that it also is an acylphosphate. However, the thiophosphoenzyme, unlike the phosphoenzyme, is acid labile at temperatures as low as 0 degree C. The acid-denatured thiophosphoenzyme has optimal stability at pH 5-6. (4) The thiophosphorylation capacity of the enzyme is equal to its phosphorylation capacity, indicating the same number of sites. Phosphorylation by ATP excludes thiophosphorylation, suggesting that the two substrates compete for the same phosphorylation site. (5) The (apparent) rate constants of thiophosphorylation (0.4 s-1 vs. 180 s-1), spontaneous dethiophosphorylation (0.04 s-1 vs. 0.5 s-1) and K+-stimulated dethiophosphorylation (0.54 s-1 vs. 230 s-1) are much lower than those for the corresponding reactions based on ATP. (6) In contrast to the phosphoenzyme, the thiophosphoenzyme is ADP-sensitive (with an apparent rate constant in ADP-induced dethiophosphorylation of 0.35 s-1, Km ADP = 48 microM at 0.1 mM ATP) and is relatively K+-insensitive. The Km for K+ in dethiophosphorylation is 0.9 mM and in dephosphorylation 0.09 mM. The thiophosphoenzyme appears to be for 75-90% in the ADP-sensitive E1-conformation.

Adenosine Diphosphate↗
PubMed1984

The H+/ATP transport ratio of the (K+ + H+)-ATPase of pig gastric membrane vesicles.

Various values have been reported for the H+/ATP transport ratio of the (K+ + H+)-ATPase of the gastric parietal cell: 4, 2 and 1. We have, therefore, reinvestigated this matter with a vesicle preparation isolated from pig gastric mucosa. The vesicles are suspended in glycylglycine buffer (pH 6.11) at 22 degrees C, and incubated until equalization of the K+ concentration inside and outside (75 mM). After addition of ATP, the initial rates of H+ uptake and ATP hydrolysis are then measured. Proton uptake is inhibited in the absence of K+ or in the presence of nigericin. The K0.5 value for proton transport is 154 microM and the Km value for ATP hydrolysis is 61 microM. The Lineweaver-Burk plot for ATP hydrolysis vs. ATP concentration is linear with a Vmax of 5.5 nmol/mg protein per s, but that for H+ uptake is not. Thus with increasing ATP concentration (6.7 to 1670 microM) the transport ratio increases from 0.3 to 1.8. Extrapolation to infinite ATP concentration gives a value of 1.89. (S.E. 0.13, N = 5) and a Hill coefficient of n = 1.21 (S.E. 0.06, N = 5) implying that the true transport ratio is 2 H+/ATP with positive cooperativity between the protons.

Adenosine Triphosphatases↗
PubMed1984

Amiloride is a cholinergic antagonist in the rabbit pancreas.

The effect of amiloride on fluid and protein secretion in the isolated rabbit pancreas and on amylase secretion in rabbit pancreatic acini has been studied. Amiloride (1 mM) has no effect on the pancreatic fluid secretion either in a normal incubation medium (143 mM Na+), or in a medium containing only 25 mM Na+. The carbachol-induced enzyme secretion is inhibited by amiloride in both systems, whereas the enzyme secretion induced by the C-terminal octapeptide of cholecystokinin ( PzO ) is not affected. Amiloride also inhibits the carbachol-induced 45Ca efflux from rabbit pancreatic acini, but again not that induced by PzO . The amiloride concentrations for half-maximal inhibition of carbachol-induced amylase secretion and 45Ca efflux are 40 and 80 microM, respectively. Amiloride also competitively inhibits the specific binding of [3H]quinuclidinyl benzylate ( [3H]QNB) to rabbit pancreatic acini, suggesting that the amiloride effect is due to competition on the level of the muscarinic acetylcholine receptor.

Acetylcholine↗
PubMed1984

Amino group modification of (Na+ + K+)-ATPase.

The effects of three amino group reagents on the activity of (Na+ + K+)-ATPase and its component K+-stimulated p-nitrophenylphosphatase activity from rabbit kidney outer medulla have been studied. All three reagents cause inactivation of the enzyme. Modification of amino groups with trinitrobenzene sulfonic acid yields kinetics of inactivation of both activities, which depend on the type and concentration of the ligands present. In the absence of added ligands, or with either Na+ of Mg2+ present, the enzyme inactivation process follows complicated kinetics. In the presence of K+, Rb+, or Tl+, protection occurs due to a change of the kinetics of inactivation toward a first-order process. ATP protects against inactivation at a much lower concentration in the absence than in the presence of Mg2+ (P50 6 microM vs. 1.2 mM). Under certain conditions (100 microM reagent, 0.2 M triethanolamine buffer, pH 8.5) modification of only 2% of the amino groups is sufficient to obtain 50% inhibition of the ATPase activity. Modification of amino groups with ethylacetimidate causes a nonspecific type of inactivation of (Na+ + K+)-ATPase. Mg2+ and K+ have no effects, and ATP only a minor effect, on the degree of modification. The K+-stimulated p-nitrophenylphosphatase activity is less inhibited than the (Na+ + K+)-ATPase activity. Half-inhibition of the (Na+ + K+)-ATPase is obtained only after 25% modification of the amino groups. Modification of amino groups with acetic anhydride also causes nonspecific inactivation of (Na+ + K+)-ATPase. Mg2+ has no effect, and ATP has only a slight protecting effect. The K+-stimulated p-nitrophenylphosphatase activity is inhibited in parallel with the (Na+ + K+)-ATPase activity. Half-inactivation of the (Na+ + K+)-ATPase activity is obtained after 20% modification of the amino groups.

4-Nitrophenylphosphatase↗