Regulation of glucagon secretion at low glucose concentrations: evidence for adenosine triphosphate-sensitive potassium channel involvement

Glucagon is a potent counterregulatory hormone that opposes the action of insulin in controlling glycemia. The cellular mechanisms by which pancreatic alpha-cell glucagon secretion occurs in response to hypoglycemia are poorly known. SUR1/K(IR)6.2-type ATP-sensitive K(+) (K(ATP)) channels have been implicated in the glucagon counterregulatory response at central and peripheral levels, but their role is not well understood. In this study, we examined hypoglycemia-induced glucagon secretion in vitro in isolated islets and in vivo using Sur1KO mice lacking neuroendocrine-type K(ATP) channels and paired wild-type (WT) controls. Sur1KO mice fed ad libitum have normal glucagon levels and mobilize hepatic glycogen in response to exogenous glucagon but exhibit a blunted glucagon response to insulin-induced hypoglycemia. Glucagon release from Sur1KO and WT islets is increased at 2.8 mmol/liter glucose and suppressed by increasing glucose concentrations. WT islets increase glucagon secretion approximately 20-fold when challenged with 0.1 mmol/liter glucose vs. approximately 2.7-fold for Sur1KO islets. Glucagon release requires Ca(2+) and is inhibited by nifedipine. Consistent with a regulatory interaction between K(ATP) channels and intra-islet zinc-insulin, WT islets exhibit an inverse correlation between beta-cell secretion and glucagon release. Glibenclamide stimulated insulin secretion and reduced glucagon release in WT islets but was without effect on secretion from Sur1KO islets. The results indicate that loss of alpha-cell K(ATP) channels uncouples glucagon release from inhibition by beta-cells and reveals a role for K(ATP) channels in the regulation of glucagon release by low glucose.     

Processive movement of single 22S dynein molecules occurs only at low ATP concentrations

We have analyzed the movement of single 22S dynein molecules from Tetrahymena cilia by using a nanometer measuring system equipped with optical tweezers. Statistical analysis proved that a single molecule of 22S dynein can move processively and develop force at low concentrations of ATP (<20 microM). The maximum force was approximately 4.7 pN, and the force-velocity curve was convex down. During force development, dynein molecules showed stepwise displacement of approximately 8 nm and frequently exhibited backward steps of approximately 8 nm. At higher concentrations of ATP (>/=20 microM) single molecules of 22S dynein were not observed to move processively. Twenty-two S dynein seems to switch over from a processive mode to a nonprocessive mode, sensing a subtle change of ATP concentrations. These observations indicate that the processivity, maximum force, and step size of dynein are similar to those of kinesin, but the ATP concentration-dependence, force-velocity relationship, and backward steps are clearly distinct from kinesin.     

Photolabeling evidence for calcium-induced conformational changes at the ATP binding site of scallop myosin.

A change in the conformation of the active site of scallop myosin under the influence of regulatory amounts of Ca2+ has been identified by use of the ADP photoaffinity analog 2-[(4-azido-2-nitrophenyl)amino]ethyl diphosphate (NANDP). NANDP, trapped at the active site with Mn2+ and vanadate, photolabeled preferentially Arg-128 of the heavy chain in the absence of added Mg2+ and Ca2+ [Kerwin, B. & Yount, R. (1992) Bioconjugate Chem. 3, 328-336]. However, addition of 2 mM Mg2+ and regulatory amounts of Ca2+ (0.01-1 microM) shifted the predominant labeling to Cys-198 of the heavy chain in a Ca(2+)-dependent manner. This Ca(2+)-dependent change in the photolabeling pattern was absent when the regulatory light chains were removed or when the unregulated head (subfragment 1) was examined under similar conditions. These results demonstrate that both Arg-128 and Cys-198 are part of the purine binding site which undergoes a conformational change in response to Ca2+ binding to the regulatory domain.     

Somatotopic organization in rat striatum: evidence for a combinational map.

2-Deoxy-D-[14C]glucose autoradiography was used in awake rats to map neural activity in the sensorimotor sector of striatum. Stimulation of hindlimb, trunk, or forelimb activated primary sensory cortex in a localized columnar pattern, indicating activation of somatosensory receptors and a discrete cortical functional unit. In sensorimotor striatum, an image analysis detection technique revealed regions of maximal activity, or features, that formed a patchy pattern of activation reminiscent of the known anatomic patterns of cortico-striate terminals. Ipsilateral as well as contralateral activation was observed. The activated areas revealed a body map in striatum that was organized in a manner consistent with cortical topography (dorsoventrally: hindlimb, trunk, forelimb) at most anteroposterior levels, similar to that found in other species. However, at other levels, a different organization (e.g., trunk, hindlimb, forelimb) was observed. Furthermore, the arrangements of body region and side were also unique at different anteroposterior levels. Thus, functional activity showed multiple, different juxtapositions of body elements--i.e., a combinational map. The data suggest that striatum may provide an anatomic substrate for different combinations of inputs necessary to select and integrate movement.     

A model for actin polymerization and the kinetic effects of ATP hydrolysis.

A model for actin polymerization is proposed in which the rate of elongation of actin filaments depends on whether adenosine 5'-triphosphate or adenosine 5'-diphosphate is bound to the two terminal subunits of the filament. This model accounts quantitatively for the experimental data on the kinetics of filament elongation and explains the effect of ATP hydrolysis on actin polymerization.     

Crystal structure of Pseudomonas aeruginosa catabolic ornithine transcarbamoylase at 3.0-A resolution: a different oligomeric organization in the transcarbamoylase family.

The crystal structure of the Glu-105-->Gly mutant of catabolic ornithine transcarbamoylase (OTCase; carbamoyl phosphate + L-ornithine = orthophosphate + L-citrulline, EC 2.1.3.3) from Pseudomonas aeruginosa has been determined at 3.0-A resolution. This mutant is blocked in the active R (relaxed) state. The structure was solved by the molecular replacement method, starting from a crude molecular model built from a trimer of the catalytic subunit of another transcarbamoylase, the extensively studied aspartate transcarbamoylase (ATCase) from Escherichia coli. This model was used to generate initial low-resolution phases at 8-A resolution, which were extended to 3-A by noncrystallographic symmetry averaging. Four phase extensions were required to obtain an electron density map of very high quality from which the final model was built. The structure, including 4020 residues, has been refined to 3-A, and the current crystallographic R value is 0.216. No solvent molecules have been added to the model. The catabolic OTCase is a dodecamer composed of four trimers organized in a tetrahedral manner. Each monomer is composed of two domains. The carbamoyl phosphate binding domain shows a strong structural homology with the equivalent ATCase part. In contrast, the other domain, mainly implicated in the binding of the second substrate (ornithine for OTCase and aspartate for ATCase) is poorly conserved. The quaternary structures of these two allosteric transcarbamoylases are quite divergent: the E. coli ATCase has pseudo-32 point-group symmetry, with six catalytic and six regulatory chains; the catabolic OTCase has 23 point-group symmetry and only catalytic chains. However, both enzymes display homotropic and heterotropic cooperativity.     

Use of a new HemoCue system for measuring haemoglobin at low concentrations

In many photometers and spectrometers used in routine practice Beer's law (relating absorbance to concentration) fails at low concentrations. A refinement of the HemoCue haemoglobin system (HemoCue AB, Sweden) provides a facility for accurate measurement of haemoglobin at the lower end of the scale. This study confirmed excellent linearity of the photometer response of the HemoCue Plasma/Low Hb photometer to haemoglobin concentration by direct measurement on whole blood samples, thus providing a reliable method when laboratory facilities are limited and where critical patient management may be dictated by the severity of anaemia. Furthermore, measurements on plasma and on samples of urine containing haemoglobin demonstrated the utility of the device for reliable quantification of plasma haemoglobin and haemoglobinuria, thus providing a useful method in investigating moderate or severe intravascular haemolysis. A study of plasma haemoglobin on stored samples of ethylenediamine tetraacetic acid (EDTA) blood showed that a small but measurable degree of lysis occurs after two days at room temperature and after four days when stored at 4 degrees C. This effect on the red cell count will reflect on the reliability of using such blood as a quality control preparation.     

Evolutionary consequences of parthogenesis: evidence from the Warramaba virgo complex.

Comparative quantitative analyses of variability in closely related parthenogenetic and sexually reproducing species have been lacking. This paper reports results of comparative analyses of relative variability carried out on the obligate thelytokous grasshopper Warramaba virgo (Key) and three closely related sexually reproducing species. Consistent patterns of differences in variability in 14 morphometric traits were found between clones and races of the parthenogenetic species which were absent in populations and species of the related sexual forms. When variability in the parthenogenetic and sexual species was compared, the parthenogenetic taxa were shown to be at least as variable as and often more variable than the sexual species.     

Direct experimental evidence for kinetic proofreading in amino acylation of tRNAIle.

Kinetic proofreading is a reaction scheme with a structure more complicated than that of Michaelis kinetics, which leads to a proofreading for errors in the recognition of a correct substrate by an enzyme. We have measured the stoichiometry between ATP hydrolysis and tRNAIle charging, using the enzyme isoleucyl-tRNA synthetase [L-isoleucine:tRNAIle ligase (AMP-forming), EC 6.1.1.5] and the amino acids isoleucine (correct) and valine (incorrect). The enzymatic deacylation of charged tRNA, which would normally prevent meaningful stoichiometry studies, was eliminated by the use of transfer factor Tu-GTP, (which binds strongly to charged tRNA) in the reaction mixture. For isoleucine, 1.5 ATP molecules are hydrolyzed per tRNA charged, but for valine, 270. These stoichiometry ratios are fundamental to kinetic proofreading, for the energy coupling is essential and proofreading is obtained only by departing from 1:1 stoichiometry between energy coupling and product formation. Within the known reaction pathway, these ratios demonstrate that kinetic proofreading induces a reduction in errors by a factor of 1/180. An overall error rate of about 10(-4) for tRNA charging is obtained by a kinetic proofreading using a fundamental discrimination level of about 10(-2), and is compatible with the low in vivo error rate of protein synthesis.     

Monitoring of low dabigatran concentrations: diagnostic performance at clinically relevant decision thresholds

Journal of Thrombosis and Thrombolysis - The direct oral anticoagulant dabigatran does not require therapeutic drug monitoring, however emergency measurements are gaining importance. Current assays...     

Rotational motion and evidence for oligomeric structures of sarcoplasmic reticulum Ca2+-activated ATPase.

The rotational motion of the sarcoplasmic reticulum Ca2+-activated ATPase (ATP phosphohydrolase, EC 3.6.1.3) has been investigated by measuring the decay of laser flash-induced dichroism with the covalently attached triplet probe eosin isothiocyanate. The Arrhenius plot for rotational mobility indicates two discontinuities at approximately 15 degrees C and approximately 35 degrees C. The experimental data are rationalized in terms of a sudden conformeric change in the ATPase at 15 degrees C and a temperature-dependent equilibrium existing between the conformationally altered ATPase and oligomeric forms of it in the temperature range 15-35 degrees C. The enzymatic activity, as indicated by a discontinuity in the Arrhenius plot for the rate of ATP hydrolysis, appears to be sensitive only to the change at 15 degrees C. There is a strong correlation between the activation energy below 15 degrees C for rotational motion (33.6 +/- 2.2 kcal/mol) and enzymatic activity (34 +/- 4 kcal/mol).     

Insulin action in aging man: evidence for tissue-specific differences at low physiologic insulin levels

This study examined the effect of age on insulin action in several tissues. Euglycemic insulin clamp studies were performed on healthy young (n = 7, 20 to 35 years, 10 and 20 mU/m2 X min insulin infusions) and old (n = 7, 66 to 80 years, 8 and 16 mU/m2 X min insulin infusions) adults. Insulin values were similar during both the lower (young, 24 +/- 1.6, old, 24 +/- 2.1 microU/ml) and higher (young, 40 +/- 3.3; old, 39 +/- 3.8 microU/ml) insulin infusion rates. Although suppression of hepatic glucose output (HGO) was more rapid (p less than .05) in the elderly group at each dose, HGO was eventually suppressed to similar levels in both age groups (low dose: young, 34.4 +/- 10.8, old, 25.3 +/- 1.8 mg/m2 X min; higher dose: young, 22.8 +/- 10.2, old, 6.2 +/- 2.1 mg/m2 X min). Glucose disposal was less (p less than .01) in the aged group at both insulin infusion rates. Suppression of C-peptide was slower in the elderly participants (p less than .05) in the low dose study. Suppression of free fatty acid and glucagon levels was the same in each age group. We concluded that the insulin resistance of aging is not generalized to all tissues.     

Spectral and kinetic evidence for the existence of two forms of bathorhodopsin.

Transient-absorption difference spectra from 320 nm to 700 nm were obtained at times ranging from 30 ns to 1200 ns after 532-nm photolysis of rhodopsin at room temperature. Kinetics on this time scale at various wavelengths are also presented. The isosbestic points between spectra acquired at successive times after photolysis shift from 510 nm to 530 nm. This shift is inconsistent with a simple process of one bathorhodopsin (BathoR) intermediate being transformed into one lumirhodopsin (LumiR) intermediate on this time scale. The kinetics at 425 nm, 515 nm, and 575 nm could not be fit well to a single-exponential expression. The data are consistent with the existence of two forms of BathoR (BathoR1 and BathoR2) that exhibit different spectra and decay kinetics. The BathoR1 absorption maximum lies near 565 nm, and the BathoR1/LumiR1 isosbestic point is near 430 nm. The BathoR2 absorption maximum lies near 535 nm, and the BathoR2/LumiR2 isosbestic point is near 480 nm. The kinetics at the isosbestic wavelengths were fit to single-exponential expressions corresponding to BathoR1 and BathoR2 lifetimes of 170 +/- 20 ns and 36 +/- 15 ns, respectively.     

Central reassessment of GH concentrations measured at local treatment centers in children with impaired growth: consequences for patient management

OBJECTIVE: GH deficiency is diagnosed in children if serum GH fails to rise above a predefined cutoff value in response to at least two stimuli. Diagnostic decisions based on this testing are highly variable between centers and depend on the GH assays used. Considering the large spectrum of commercially available GH assays, we wanted to evaluate the agreement between assays, and to test whether assay-related variability of diagnostic decisions could be reduced by reassessment of peak GH concentrations in a reference center. DESIGN: We reanalysed 699 peak GH serum samples obtained after GH testing of 382 children and adolescents from 19 centers using three reference assays and compared these results with those obtained with the local assays. A subgroup of 132 patients tested with the combination of insulin hypoglycemia test and arginine test was evaluated for changes in the assignment to the diagnostic group of GH deficiency. RESULTS: The mean difference between methods ranged from 5.4 to 10.3 mU/l, slopes of the regression lines from 1.28 to 1.65. Significant non-linearity was detected in five of six assay comparisons, indicating that most assay results cannot be interconverted by the use of a factor. Overall agreement between reference and local assays was only moderate. Significant changes in diagnostic assignment occurred when different assays were used on the same patient (P<0.0001-P<0.0023). Based on GH remeasurement by one reference assay, 36 of 132 patients were categorized differently, with 35 patients changing into the GH-deficient group. Similar findings were obtained with the other reference assays. CONCLUSIONS: To decrease variability in GH testing related to assays and cutoff values, we recommend nationwide reassessment of GH peak sera in reference centers. Decisions to treat GH deficiency should incorporate the reference center results.     

Mechanism of mitotic block and inhibition of cell proliferation by taxol at low concentrations.

Taxol inhibited HeLa cell proliferation by inducing a sustained mitotic block at the metaphase/anaphase boundary. Half-maximal inhibition of cell proliferation occurred at 8 nM taxol, and mitosis was half-maximally blocked at 8 nM taxol. Inhibition of mitosis was associated with formation of an incomplete metaphase plate of chromosomes and an altered arrangement of spindle microtubules that strongly resembled the abnormal organization that occurs with low concentrations of vinblastine and other antimitotic compounds. No increase in microtubule polymer mass occurred below 10 nM taxol. The mass of microtubules increased half-maximally at 80 nM taxol and attained maximal levels (5 times normal) at 330 nM taxol. At submicromolar concentrations, taxol suppressed growing and shortening at the ends of microtubules reassembled in vitro from bovine brain tubulin in a manner that resembled suppression by vinblastine. Taxol was concentrated in HeLa cells several hundredfold to levels that were similar to those which suppressed dynamic instability in vitro. The results indicate that taxol shares a common antiproliferative mechanism with vinblastine. At its lowest effective concentrations, taxol appears to block mitosis by kinetically stabilizing spindle microtubules and not by changing the mass of polymerized microtubules.     

Experimental evidence for kinetic proofreading in the aminoacylation of tRNA by synthetase.

The enzymatic aminoacylation of tRNA can be viewed as a means of proofreading either the amino acid or the tRNA or both. We have conducted further experimental tests of kinetic proofreading in discriminating between cognate and noncognate amino acids and tRNAs as follows: (formula: see text). In cases (i) and (ii) the amino acids are proofread, in cases (iii) and (iv) the tRNA is proofread, and in case (v), both the amino acid and the tRNA are proofread. ATP consumed per acylation was 400, 1.5, 40, 25, and 1000, respectively. High ATP/aminoacylation ratios are diagnostic for kinetic proofreading.     

Mechanisms of flow-mediated signal transduction in endothelial cells: kinetics of ATP surface concentrations.

Intracellular free calcium ([Ca2+]i) was measured in single cells of a confluent endothelial monolayer subjected to defined flow. Flow medium containing adenosine triphosphate (ATP) was used to study the influence of flow forces upon agonist-response coupling as mediated via the P2y-purinoceptor. [Ca2+]i responses were highly sensitive to the fluid motion at the cell surface; consecutive small increases of flow stimulated large [Ca2+]i transients with the levels returning to baseline at the new flow rate within 250 s. The characteristics of [Ca2+]i transients were also influenced by decreasing flow. Since potent ectonucleotidases at the endothelial cell surface rapidly degrade ATP, we postulated that a combination of flow and degradative enzymes regulates the mass transport of ATP in the boundary layer. The hypothesis predicts that step increases of flow exceed the capacity of the ectonucleotidases and allow ATP to reach the receptor. Experiments were conducted to compare ATP and ADP beta S, a nonhydrolyzable ATP analog that resists degradation by surface ectonucleotidases, and calculations of ATP mass transport to the cell surface were compared to estimates of surface clearance rates. Calculations of mass transport coefficients for ATP in the boundary layer demonstrated that changes of flow which elicited a prominent [Ca2+]i response represented 26-73% changes in the mass transport of ATP from the bulk fluid. When steadystate mass transport coefficients for ATP under various flow conditions were compared with the estimated rate constant for surface degradation of ATP, ratios close to unity were obtained. These results suggest that both boundary layer mass transport and ATP clearance rates can be rate-limiting for flow-mediated activation of the P2y-receptor. The experiments provide evidence for differential signal transduction responses in the endothelium driven by diffusion gradients (derived from both the blood and the vessel wall), which are likely to vary widely in the complex flow fields encountered in vivo.     

Heparin-catalyzed inhibitor/protease reactions: kinetic evidence for a common mechanism of action of heparin.

Three different heparin-catalyzed inhibitor/protease reactions were studied: antithrombin III/thrombin, heparin cofactor II/thrombin, antithrombin III/factor Xa. The three reactions were saturable with respect to both inhibitor and protease. The initial reaction velocity, for each reaction, could be described by the general rate equation for a random-order bireactant enzyme-catalyzed reaction. The kinetic parameters for the heparin-catalyzed antithrombin III/thrombin and antithrombin III/factor Xa reactions differed in terms of apparent maximum velocity (Vmax) and apparent heparin-protease dissociation constant values. The apparent heparin-antithrombin III dissociation constant values were the same for both reactions. The kinetic parameters for the heparin-catalyzed antithrombin III/thrombin and heparin cofactor II/thrombin reactions differed in terms of apparent Vmax and apparent heparin-inhibitor dissociation constant values. The apparent heparin-thrombin dissociation constant values were the same for both reactions. The results are consistent with a general mechanism of action of heparin for the three reactions that, in its simplest form, requires only that both protease and inhibitor bind to heparin for catalysis to occur.     

Sex hormone status of male patients with rheumatoid arthritis: evidence of low serum concentrations of testosterone at baseline and after human chorionic gonadotropin stimulation

Serum concentrations of luteinizing hormone, follicle-stimulating hormone, prolactin, 17 beta-estradiol, testosterone, androstenedione, dehydrotestosterone, dehydroepiandrosterone sulfate, and cortisol were examined in 14 men with rheumatoid arthritis (RA) and in age-matched osteoarthritis controls. Hypophyseal, adrenal, and testicular responses to stimulation with luteinizing hormone-releasing hormone, adrenocorticotropin, and human chorionic gonadotropin, respectively, were evaluated in 8 RA patients and in 8 age-matched healthy volunteers. Basal serum testosterone concentrations were significantly lower in male RA patients than in the osteoarthritis control subjects (P less than 0.01). After human chorionic gonadotropin stimulation, serum concentrations of testosterone were also lower in the RA patients than in normal healthy controls (P less than 0.05). These findings suggest that diminished testicular steroid biosynthesis might contribute to the serum testosterone deficiency observed in male RA patients.