Caveolae and propofol effects on airway smooth muscle.

BACKGROUND: The i.v. anaesthetic propofol produces bronchodilatation. Airway relaxation involves reduced intracellular Ca(2+) ([Ca(2+)](i)) in airway smooth muscle (ASM) and lipid rafts (caveolae), and constitutional caveolin proteins regulate [Ca(2+)](i). We postulated that propofol-induced bronchodilatation involves caveolar disruption. METHODS: Caveolar fractions of human ASM cells were tested for propofol content. [Ca(2+)](i) responses of ASM cells loaded with fura-2 were performed in the presence of 10 µM histamine with and without clinically relevant concentrations of propofol (10 and 30 µM and intralipid control). Effects on sarcoplasmic reticulum (SR) Ca(2+) release were evaluated in zero extracellular Ca(2+) using the blockers Xestospongin C and ryanodine. Store-operated Ca(2+) entry (SOCE) after SR depletion was evaluated using established techniques. The role of caveolin-1 in the effect of propofol was tested using small interference RNA (siRNA) suppression. Changes in intracellular signalling cascades relevant to [Ca(2+)](i) and force regulation were also evaluated. RESULTS: Propofol was present in ASM caveolar fractions in substantial concentrations. Exposure to 10 or 30 µM propofol form decreased [Ca(2+)](i) peak (but not plateau) responses to histamine by ~40%, an effect persistent in zero extracellular Ca(2+). Propofol effects were absent in caveolin-1 siRNA-transfected cells. Inhibition of ryanodine receptors prevented propofol effects on [Ca(2+)](i), while propofol blunted [Ca(2+)](i) responses to caffeine. Propofol reduced SOCE, an effect also prevented by caveolin-1 siRNA. Propofol effects were associated with decreased caveolin-1 expression and extracellular signal-regulated kinase phosphorylation. CONCLUSIONS: These novel data suggest a role for caveolae (specifically caveolin-1) in propofol-induced bronchodilatation. Due to its lipid nature, propofol may transiently disrupt caveolar regulation, thus altering ASM [Ca(2+)](i).

Protein dynamics in whole body and in splanchnic and leg tissues in type I diabetic patients.

To elucidate the mechanism of insulin's anticatabolic effect in humans, protein dynamics were evaluated in the whole-body, splanchnic, and leg tissues in six C-peptide-negative type I diabetic male patients in the insulin-deprived and insulin-treated states using two separate amino acid models (leucine and phenylalanine). L-(1-13C,15N)leucine, L-(ring-2H5)phenylalanine, and L-(ring-2H2) tyrosine were infused intravenously, and isotopic enrichments of [1-13C,15N]-leucine, (13C)leucine, (13C)ketoisocaproate, (2H5)phenylalanine, [2H4]tyrosine, (2H2)tyrosine, and 13CO2 were measured in arterial, hepatic vein, and femoral vein samples. Whole-body leucine flux, phenylalanine flux, and tyrosine flux were decreased (< 0.01) by insulin treatment, indicating an inhibition of protein breakdown. Moreover, insulin decreased (< 0.05) the rates of leucine oxidation and leucine transamination (P < 0.01), but the percent rate of ketoisocaproate oxidation was increased by insulin (P < 0.01). Insulin also reduced (< 0.01) whole-body protein synthesis estimated from both the leucine model (nonoxidative leucine disposal) and the phenylalanine model (disposal of phenylalanine not accounted by its conversion to tyrosine). Regional studies demonstrated that changes in whole body protein breakdown are accounted for by changes in both splanchnic and leg tissues. The changes in whole-body protein synthesis were not associated with changes in skeletal muscle (leg) protein synthesis but could be accounted for by the splanchnic region. We conclude that though insulin decreases whole-body protein breakdown in patients with type I diabetes by inhibition of protein breakdown in splanchnic and leg tissues, it selectively decreases protein synthesis in splanchnic tissues, which accounted for the observed decrease in whole-body protein synthesis. Insulin also augmented anabolism by decreasing leucine transamination.

Differential regulation of amino acid exchange and protein dynamics across splanchnic and skeletal muscle beds by insulin in healthy human subjects.

To define the mechanism of insulin's anticatabolic action, the effects of three different dosages of insulin (0.25, 0.5, and 1.0 mU x kg(-1) x min(-1)) versus saline on protein dynamics across splanchnic and skeletal muscle (leg) beds were determined using stable isotopes of phenylalanine, tyrosine, and leucine in 24 healthy subjects. After an overnight fast, protein breakdown in muscle exceeded protein synthesis, causing a net release of amino acids from muscle bed, while in the splanchnic bed protein synthesis exceeded protein breakdown, resulting in a net uptake of these amino acids. Insulin decreased (P < 0.003) muscle protein breakdown in a dose-dependent manner with no effect on muscle protein synthesis, thus decreasing the net amino acid release from the muscle bed. In contrast, insulin decreased protein synthesis (P < 0.03) in the splanchnic region with no effect on protein breakdown, thereby decreasing the net uptake of the amino acids. In addition, insulin also decreased (P < 0.001) leucine nitrogen flux substantially more than leucine carbon flux, indicating increased leucine transamination (an important biochemical process for nitrogen transfer between amino acids and across the organs), in a dose-dependent manner, with the magnitude of effect being greater on skeletal muscle than on the splanchnic bed. In conclusion, muscle is in a catabolic state in human subjects after an overnight fast and provides amino acids for synthesis of essential proteins in the splanchnic bed. Insulin achieves amino acid balance across splanchnic and skeletal muscle beds through its differential effects on protein dynamics in these tissue beds.

Assessment of postabsorptive renal glucose metabolism in humans with multiple glucose tracers.

The contribution of the kidneys to postabsorptive endogenous glucose production is a matter of controversy. To assess whether this could relate to the use of various isotopical methods with different analytical performance capabilities, we measured glucose kinetics in 12 healthy subjects. Blood samples were taken from the femoral artery and the renal vein after 4 h of [6,6-2H2]glucose infusion (for gas chromatography [GC]/mass spectrometry [MS] analysis), and renal plasma flow was determined with paraaminohippurate. In addition, six subjects received uniformly labeled [13C]glucose (for GC/combustion/isotope ratio MS [IRMS]) and [3-3H]glucose (for counting of radioactive disintegrations). Arterial glucose concentrations (means +/- SD) were 4.2+/-0.1 mmol/l, and endogenous glucose production rates using [2H2]glucose were 2.2+/-0.1 mg x kg(-1) x min(-1) or 818+/-50 micromol/min. Dilution of [2H2]glucose across the kidney was 0.79+/-1.32%, and renal glucose production (RGP) rates were 27+/-72 micromol/min. In the six subjects receiving additional tracers, dilutions across the kidney were 2.83+/-0.72 and 0.54+/-1.20 (for [U-13C]glucose and [3-3H]glucose, respectively, the dilution with [U-13C] being higher than that with [2H2] (P = 0.007). Corresponding RGP values were 144+/-39 and 43+/-76 micromol/min for [U-13C] and [3-3H], respectively. In conclusion, we found that the highly sensitive [U-13C] GC/Combustion/IRMS technique showed consistent dilution of label across the kidney, whereas the less sensitive techniques gave some negative values and smaller RGP rates. Thus, depending on which technique is being used, a fivefold difference in calculated RGP values may be encountered. The methodological variability of our data suggests that extrapolation from regional renal measurements to the whole-body level should be perfumed with caution.

Molecular size and symmetry of the bacterioferritin of Escherichia coli. X-ray crystallographic characterization of four crystal forms.

X-ray crystallographic data from four crystal forms of Escherichia coli bacterioferritin show that the molecule has a diameter in the range 119 to 128 A. Molecules are composed of 24 subunits arranged in 432 symmetry. In both size and symmetry the molecule resembles ferritin from eukaryotes. The four crystal forms are monoclinic, space group P2(1) with unit cell dimensions a = 118.7 A, b = 211.6 A, c = 123.3 A and beta = 119.1 degrees; orthorhombic, C222(1), a = 128.7 A, b = 197.1 A, c = 202.8 A; tetragonal, P4(2)2(1)2, a = b = 210.6 A, c = 145.0 A and cubic, I432, a = 146.9 A.

Mechanism of action of aspartate aminotransferase proposed on the basis of its spatial structure.

Aspartate aminotransferase is a pyridoxal phosphate-dependent enzyme that catalyses the transamination reaction: L-aspartate + 2-oxoglutarate----oxaloacetate + L-glutamate. The enzyme shuttles between its pyridoxal and pyridoxamine forms in a double-displacement process. This paper proposes a mechanism of action that delineates the dynamic role of the protein moiety of this enzyme. It is based on crystallographically determined spatial structures (at 2.8 A resolution) of the mitochondrial isoenzyme in its unliganded forms and in complexes with substrate analogues, as well as on model building studies. The enzyme is composed of two identical subunits, which consist of two domains. The coenzyme is bound to the larger domain and is situated in a pocket near the subunit interface. The proximal and distal carboxylate group of dicarboxylic substrates are bound to Arg386 and Arg292 , respectively, the latter residue belonging to the adjacent subunit. These interactions largely determine the substrate specificity of the enzyme. They not only position the substrate efficient catalysis but also bring about a bulk movement of the small domain that closes the active site crevice and moves Arg386 about 3 A closer to the coenzyme. The replacement of the epsilon-amino group of Lys258 by the alpha-amino group of the substrate in the aldimine bond to pyridoxal phosphate is accompanied by a tilting of the coenzyme by approximately 30 degrees. The released epsilon-amino group of Lys258 serves as a proton acceptor/donor in the 1,3- prototropic shift producing the ketimine intermediate. At this stage, or after hydrolysis of the ketimine bond, the coenzyme rotates back to an orientation between that in the "external" aldimine intermediate and that in the pyridoxal form. Throughout this process, the protonated pyridine nitrogen atom maintains a hydrogen bond to the beta-carboxylate group of Asp222 . Upon formation of the pyridoxamine form, the small domain moves back to its original position. The proposed mechanism is compatible with the known kinetic and stereochemical features of enzymic transamination.

Comparison of the three-dimensional structures of recombinant human H and horse L ferritins at high resolution.

Mammalian ferritins are 24-mers assembled from two types of polypeptide chain which provide the molecule with different functions. H(eavy) chains catalyse the first step in iron storage, the oxidation of iron(II). L(ight) chains promote the nucleation of the mineral ferrihydrite enabling storage of iron(III) inside the protein shell. We report here the comparison of the three-dimensional structures of recombinant human H chain (HuHF) and horse L chain (HoLF) ferritin homopolymers, which have been refined at 1.9 A resolution. There is 53% sequence identity between these molecules, and the two structures are very similar, the H and L subunit alpha-carbons superposing to within 0.5 A rms deviation with 41 water molecules in common. Nevertheless, there are significant important differences which can be related to differences in function. In particular, the centres of the four-helix bundles contain distinctive groups of hydrophilic residues which have been associated with ferroxidase activity in H chains and enhanced stability in L chains. L chains contain a group of glutamates associated with mineralisation within the iron storage cavity of the protein.

Structural homology between mouse liver and horse spleen ferritins.

Mouse liver ferritin is composed almost exclusively of polypeptide chains similar in molecular mass (22 kDa) to that characteristic of the major chain (H) found in heart ferritin isolated from human, horse or rat. In these species the predominant polypeptide of liver (L) is smaller (about 20 kDa). Here we show that mouse liver and horse spleen ferritins and apoferritins exhibit extensive structural homology as judged by the similarity in the diffraction patterns of their crystals grown from cadmium sulphate solutions. Implications of this finding are discussed.

Effect of intravenous insulin treatment on in vivo whole body leucine kinetics and oxygen consumption in insulin-deprived type I diabetic patients.

In vivo leucine metabolism was studied after an overnight fast in nine type I diabetic patients and nine healthy control subjects using L-[1-13C] leucine as a tracer. In the insulin-deprived state, leucine flux (reflecting proteolysis), leucine oxidation, and plasma leucine concentrations were higher in the diabetic patients than in the control subjects (P less than .001). In 4 of the 9 insulin-deprived diabetic patients, a four-hour intravenous insulin treatment decreased plasma glucose and leucine concentrations and leucine flux, but failed to decrease leucine oxidation. In the remaining 5 of the 9 diabetic patients, uninterrupted insulin treatment prior to the study and a seven-hour intravenous insulin treatment during the study period decreased not only the concentrations of plasma glucose and leucine and leucine flux, but also leucine oxidation (P less than .01). In all 9 diabetic patients the nonoxidative portion of leucine flux (reflecting protein synthesis) decreased during insulin treatment (P less than .01), but this decrease was lower than that of leucine flux (reflecting proteolysis), and therefore protein was conserved during insulin treatment. We conclude that the effect of insulin on proteolysis (reflected by leucine flux) is more rapid than its effect on leucine oxidation, but on aggressive insulin treatment accelerated leucine oxidation also was decreased in type I diabetic patients.

Sources of propionate in inborn errors of propionate metabolism.

Amino acids are widely regarded as the most important sources of propionate in disorders of propionate metabolism. Propionate production was measured in the fasting state by continuous infusion of sodium [1-13C]propionate in three children with methylmalonic acidemia (MMA) and three with propionic acidemia (PA). The contribution of isoleucine, valine, threonine, and methionine catabolism to total propionate production was estimated by extrapolation from the hydroxylation of phenylalanine determined by a continuous-infusion [2H5]phenylalanine technique. The contribution of gut bacterial propionate production was determined by measuring total propionate production before and after treatment with oral metronidazole (10 to 20 mg/kg/d for 1 week). Amino acid catabolism accounted for a mean of 51.7% (range, 24.5% to 66.4%) of total propionate production. The mean decrease in propionate production after metronidazole was 22.2% +/- 8.5 (P less than .02); this percentage is likely to represent the minimum propionate production attributable to gut bacteria. Approximately 30% of total propionate production was unaccounted for, and is likely to arise primarily from odd-chain fatty acid catabolism in the fasting state. These results indicate that sources of propionate other than from protein catabolism are important in disorders of propionate metabolism, and explain the generally disappointing response to dietary protein restriction.

Calculation of turnover rates in stable-isotope studies.

Tserng and Kalhan have raised the question of the appropriate equation to use for the measurement of turnover rate in a stable-isotope study. In a comparison of glucose turnover measured with 2H-glucose and with 13C-glucose they used five apparently different equations and obtained conflicting answers. There is, however, no difference in principle between the use of a stable isotope as a tracer and the use of a radioactive isotope, and the rate of appearance of tracee in a steady-state system (the turnover) can therefore be shown to be proportional to the equilibrium dilution of the infused tracer. Because the sensitivity of measurement of this dilution made using a gas chromatograph-mass spectrometer is lower than that made by radioactivity measurement, the contribution to the measured turnover rate due to the infusate cannot be neglected, as it usually is in radioisotope work. A convenient calibration curve to establish this dilution is the mole ratio of the pure infusate against the area ratio for the relevant ions. Tserng and Kalhan's apparently conflicting results for glucose turnover using 13C-glucose as the tracer can all be shown to amount to approximately 11.6 mumol min-1 kg-1. This value is only slightly lower (0.05 less than P less than 0.1) than that obtained using 2H-glucose as the tracer and supports the use of 13C-glucose as an alternative.

Decompression sickness presenting as forearm swelling and peripheral neuropathy: a case report.

Neurological symptoms after decompression from a dive are usually thought to be manifestations of central nervous system (CNS) decompression sickness (DCS). We present a case of DCS in which neurological symptoms are present but which the clinical findings, magnetic resonance imaging and electroneuromyographic studies suggest were caused by muscle injury and exacerbation of an existing peripheral neuropathy. This finding supports the alternative hypothesis that some neurological symptoms and signs in DCS are due to effects on peripheral nerves rather than the CNS.

Propantheline bromide plasma level, urinary excretion and pharmacological data in a comparison of the bioavailability of three oral formulations of Pro-Banthine.

To determine the comparative bioavailability of three oral formulations of propantheline bromide (PB) by both pharmacokinetic and pharmacodynamic parameters, six normal men received three standard Pro-Banthine 15 mg tablets, two prolonged acting (PA) Pro-Banthine 30 mg tablets or one developmental PA Pro-Banthine 45 mg capsule, in a study of balanced random crossover design. Plasma concentrations and urinary excretion of the unchanged drug were measured after each treatment using a stable isotope dilution assay. Salivary secretion rate and heart rate measurements were also made at intervals after each medication. The standard Pro-Banthine formulation was significantly more bioavailable, weight for weight, than either the developmental PA capsule (45 mg), p less than 0.05, or the two 30 mg PA tablets (60 mg), p less than 0.01, based on urinary excretion and plasma levels of PB and on salivary secretion and heart rate data. There was no evidence of significant prolonged action for the PA formulations.