Identification of amino acid residues that determine pH dependence of ligand binding to the asialoglycoprotein receptor during endocytosis.

The rat hepatic asialoglycoprotein receptor mediates clearance of galactose- and N-acetylgalactosamine-terminated glycoproteins by endocytosis, binding ligands through a C-type, Ca(2+)-dependent carbohydrate-recognition domain (CRD) at extracellular pH and releasing them at lower pH in endosomes. At physiological Ca(2+) concentrations, the midpoint for ligand release from the CRD of the major subunit of the receptor is pH 7.1. In contrast, the midpoint is pH 5.0 for a galactose-binding derivative of the homologous C-type CRD of serum mannose-binding protein, which would thus not efficiently release ligand at an endosomal pH of 5.4. Site-directed mutagenesis of the CRD from the major subunit of the asialoglycoprotein receptor has been used to identify residues that are essential for efficient release of ligand at endosomal pH. The effects of changes to residues His(256), Asp(266), and Arg(270) singly and in combination indicate that these residues reduce the affinity of the CRD for Ca(2+), so that ligands are released at physiological Ca(2+) concentrations. The proximity of these three residues to the ligand-binding site at Ca(2+) site 2 of the domain suggests that they form a pH-sensitive switch for Ca(2+) and ligand binding. Introduction of histidine and aspartic acid residues into the mannose-binding protein CRD at positions equivalent to His(256) and Asp(266) raises the pH for half-maximal binding of ligand to 6.1. The results, as well as sequence comparisons with other C-type CRDs, confirm the importance of these residues in conferring appropriate pH dependence in this family of domains.

Cervical magnetic stimulation of the phrenic nerves in bilateral diaphragm paralysis.

Cervical magnetic stimulation (CMS) produces a greater twitch transdiaphragmatic pressure (TwPdi) than electrical stimulation. This may be because CMS produces rib cage muscle activation, thus producing an inspiratory action independent of the diaphragm. Alternatively, CMS could merely stiffen the rib cage, allowing the diaphragm to act efficiently, by contracting against a stable rib cage. To examine these two hypotheses we studied five patients with isolated bilateral diaphragm paralysis using CMS and bilateral electrical phrenic stimulation (BES). TwPdi, twitch esophageal pressure (TwPes), and twitch gastric pressure (TwPgas) were measured. We also assessed maximal sniff esophageal and transdiaphragmatic pressures (SnPes) (SnPdi), maximal inspiratory and expiratory mouth pressures (MIP) (MEP), and fall in VC on moving from an upright to a supine position. Respiratory muscle strength tests were consistent with bilateral diaphragm paralysis, and the MEPs confirmed normal expiratory muscle function. The patients were able to generate a mean SnPes of -30 cm H2O, mainly because of inspiratory activity of rib cage and neck muscles. However, TwPdi and TwPes during both CMS and BES were close to zero. We conclude that in our patients with diaphragm paralysis caused by neuralgic amyotrophy, CMS stiffens the rib cage but does not have an inspiratory action independent of the diaphragm.

Identification of essential histidine residues in UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase-T1.

UDP-N-acetyl-d-galactosamine:polypeptide N-acetylgalactosaminyltransferases (ppGaNTases) catalyse the initial step of mucin-type O-glycosylation. The activity of bovine ppGaNTase-T1 isoenzyme was inhibited by diethyl pyrocarbonate (DEPC) modification. Activity was partially restored by hydroxylamine treatment, indicating that one of the reactive residues was a histidine. The transferase was protected against DEPC inactivation when UDP-GalNAc and EPO-G, a peptide pseudo-substrate PPDAAGAAPLR, were simultaneously present, while presence of EPO-G alone did not alter DEPC inactivation. However, inclusion of UDP-GalNAc alone potentiated DEPC-inhibition of the enzyme, suggesting that UDP-GalNAc binding changes the accessibility or reactivity of an essential histidine residue. Deletion of the first 56 amino acids (including one hisitidine residue) yielded a fully active secreted form of the bovine ppGaNTase-T1 enzyme. Each of the 14 remaining histidines in the enzyme were mutated to alanine, and the recombinant mutants were recovered from COS7 cells. The mutation of histidine residues His211-->Ala and His344-->Ala resulted in recombinant proteins with no detectable enzymic activity. A significant decrease in the initial rate of GalNAc transfer to the substrate was observed with mutants His125-->Ala and His341-->Ala (1% and 6% of wild-type activity respectively). Mutation of the remaining ten histidine residues yielded mutants that were indistinguishable from the wild-type enzyme. Mutagenesis and SDS/PAGE analysis of all N-glycosylation sequons revealed that positions N-95 and N-552 are occupied by N-linked sugars in COS7 cells. Ablation of either site did not perturb enzyme biosynthesis or enzyme activity.

Clinical assessment of diaphragm strength by cervical magnetic stimulation of the phrenic nerves.

BACKGROUND: Accurate assessment of diaphragm strength can be difficult. Transdiaphragmatic pressure (PDI) measurements during volitional manoeuvres are useful but it may be difficult to ensure maximum patient effort. Magnetic stimulation of the phrenic nerves is easy to perform and the results are reproducible in normal subjects. The purpose of the present study was to evaluate the usefulness of magnetic stimulation of the phrenic nerves in the assessment of diaphragm weakness in patients. METHODS: Sixty-six patients referred for assessment of respiratory muscle strength and 23 normal subjects were studied. Twitch PDI (TwPDI) following magnetic stimulation of the phrenic nerves and sniffPDI were obtained in all individuals. TWPDI following bilateral electrical stimulation of the phrenic nerves was also obtained in eight patients. RESULTS: Mean (SD) TwPdi for the normal subjects was 31 (6) cm H2O and 18 (11) cm H2O for the patients. TwPDI and sniffPDI were correlated (r = 0.77). Seven of the 37 patients (19%) with a reduced sniffPDI had a TwPDI within the normal range whereas two of the 32 patients (6%) with a reduced TwPDI had a normal sniffPDI. TwPDI was similar with magnetic and electrical stimulation. CONCLUSIONS: TwPDI following magnetic stimulation of the phrenic nerves is a clinically useful measurement when assessing diaphragm weakness.

Effect of maximum ventilation on abdominal muscle relaxation rate.

BACKGROUND: When the demand placed on the respiratory system is increased, the abdominal muscles become vigorously active to achieve expiration and facilitate subsequent inspiration. Abdominal muscle function could limit ventilatory capacity and a method to detect abdominal muscle fatigue would be of value. The maximum relaxation rate (MRR) of skeletal muscle has been used as an early index of the onset of the fatiguing process and precedes failure of force generation. The aim of this study was to measure MRR of abdominal muscles and to investigate whether it slows after maximum isocapnic ventilation (MIV). METHODS: Five normal subjects were studied. Each performed short sharp expiratory efforts against a 3 mm orifice before and immediately after a two minute MIV. Gastric pressure (PGA) was recorded and MRR (% pressure fall/10 ms) for each PGA trace was determined. RESULTS: Before MIV the mean (SD) maximum PGA MRR for the five subjects was 7.1 (0.8)% peak pressure fall/10 ms. Following MIV mean PGA MRR was decreased by 30% (range 25-35%), returning to control values within 5-10 minutes. CONCLUSIONS: The MRR of the abdominal muscles, measured from PGA, is numerically similar to that described for the diaphragm and other skeletal muscles. After two minutes of maximal isocapnic ventilation abdominal muscle MRR slows, indicating that these muscles are sufficiently heavily loaded to initiate the fatiguing process.

Diaphragm fatigue following maximal ventilation in man.

When highly motivated normal subjects perform maximal isocapnic ventilation, a substantial fall in ventilation is observed during the first minute associated with slowing of the maximum relaxation rate (MRR) of the inspiratory muscles. This suggests that these muscles are excessively loaded, raising the possibility that overt contractile failure of the diaphragm contributes to the fall in ventilation. We therefore investigated the effect of maximal isocapnic ventilation (MIV) on twitch transdiaphragmatic pressure (Pdi,Tw) elicited by cervical magnetic stimulation. We measured Pdi,Tw before and after 2 min MIV in nine normal subjects. Initial mean (SD) ventilation for the nine subjects was 196 (15) L.min-1 falling by 35% at 1 min. Pdi,Tw fell following MIV, at 10 min was reduced by 24%, and remained substantially reduced 90 min after MIV. No change in Pdi,Tw was observed during control studies in which subjects were studied with the same protocol but omitting MIV. We conclude that diaphragmatic contractility is reduced after 2 min maximal isocapnic ventilation and diaphragmatic fatigue may be a limiting factor in maximal ventilation in man.

Unilateral magnetic stimulation of the phrenic nerve.

BACKGROUND: Electrical stimulation of the phrenic nerve is a useful non-volitional method of assessing diaphragm contractility. During the assessment of hemidiaphragm contractility with electrical stimulation, low twitch transdiaphragmatic pressures may result from difficulty in locating and stimulating the phrenic nerve. Cervical magnetic stimulation overcomes some of these problems, but this technique may not be absolutely specific and does not allow the contractility of one hemidiaphragm to be assessed. This study assesses both the best means of producing supramaximal unilateral magnetic phrenic stimulation and its reproducibility. This technique is then applied to patients. METHODS: The ability of four different magnetic coils to produce unilateral phrenic stimulation in five normal subjects was assessed from twitch transdiaphragmatic pressure (TwPDI) measurements and diaphragmatic electromyogram (EMG) recordings. The results from magnetic stimulation were compared with those from electrical stimulation. To determine whether the magnetic field affects the contralateral phrenic nerve as well as the intended phrenic nerve, EMG recordings from each hemidiaphragm were compared during stimulation on the same side and the opposite side relative to the recording electrodes. The EMG recordings were made from skin surface electrodes in five normal subjects and from needle electrodes placed in the diaphragm during cardiac surgery in six patients. Similarly, the direction of hemidiaphragm movement was evaluated by ultrasonography. To determine the usefulness of the technique in patients the 43 mm mean diameter double coil was used in 54 patients referred for assessment of possible respiratory muscle weakness. These results were compared with unilateral electrical phrenic stimulation, maximum sniff PDI, and TwPDI during cervical magnetic stimulation. RESULTS: In the five normal subjects supramaximal stimulation was established for eight out of 10 phrenic nerves with the 43 mm double coil. Supramaximal unilateral magnetic stimulation produced a higher TwPDI than electrical stimulation (mean (SD) 13.4 (2.5) cm H2O with 35 mm coil; 14.1 (3.8) cm H2O with 43 mm coil; 10.0 (1.7) cm H2O with electrical stimulation). Spread of the magnetic field to the opposite phrenic nerve produced a small amplitude contralateral diaphragm EMG measured from skin surface electrodes which reached a mean of 15% of the maximum EMG amplitude produced by ipsilateral stimulation. Similarly, in six patients with EMG activity recorded directly from needle electrodes, the contralateral spread of the magnetic field produced EMG activity up to a mean of 3% and a maximum of 6% of that seen with ipsilateral stimulation. Unilateral magnetic stimulation of the phrenic nerve was rapidly achieved and well tolerated. In the 54 patients unilateral magnetic TwPDI was more closely related than unilateral electrical TwPDI to transdiaphragmatic pressure produced during maximum sniffs and cervical magnetic stimulation. Unilateral magnetic stimulation eliminated the problem of producing a falsely low TwPDI because of technical difficulties in locating and adequately stimulating the nerve. Eight patients with unilateral phrenic nerve paresis, as indicated by a unilaterally elevated hemidiaphragm on a chest radiograph and maximum sniff PDI consistent with hemidiaphragm weakness, were all accurately identified by unilateral magnetic stimulation. CONCLUSIONS: Unilateral magnetic phrenic nerve stimulation is easy to apply and is a reproducible technique in the assessment of hemidiaphragm contractility. It is well tolerated and allows hemidiaphragm contractility to be rapidly and reliably assessed because precise positioning of the coils is not necessary. This may be particularly useful in patients. In addition, the anterolateral positioning of the coil allows the use of the magnet in the supine patient such as in the operating theatre or intensive care unit.

The effect of lung volume on transdiaphragmatic pressure.

Diaphragm strength can be assessed by measurement of transdiaphragmatic pressure (Pdi) in response to stimulation of the phrenic nerves. The length-tension relationship of the diaphragm can be studied by measuring twitch Pdi over the range of lung volume. Previous studies of the relationship between lung volume and diaphragm strength have used the technique of electrical stimulation of the phrenic nerves. In these studies, the phenomenon of twitch potentiation has not been taken into account. It has previously been shown that prior contraction of the diaphragm can greatly enhance the twitch response, thus affecting the measurements. The aim of this study was to investigate the relationship between unpotentiated twitch Pdi and lung volume for volumes ranging from residual volume (RV) to total lung capacity (TLC) in normal subjects. Great care was taken to avoid muscle potentiation. For this purpose, we stimulated the phrenic nerves with a magnetic stimulator. In addition, we used positive pressure to inflate the lungs to high lung volumes. The impact of twitch potentiation on the length-tension relationship was investigated by subjects making maximum inspiratory efforts prior to phrenic nerve stimulation. The unpotentiated twitch Pdi decreased in a linear fashion with increasing lung volume over the full range of vital capacity by 0.54 kPa.L-1. Potentiation increased twitch Pdi by 40% at FRC and the effect was similar, in absolute terms, at all lung volumes. In relative terms, the effect of potentiation became greater as lung volume increased, and more than doubled twitch Pdi at TLC. With increasing lung volume, there is a linear fall in unpotentiated twitch Pdi with a slope that is less steep, over the same range of absolute lung volume, than previously reported. When assessing diaphragm strength by the twitch technique, it is essential to control for lung volume and equally important to control for twitch potentiation.

Kinetic analysis of a recombinant UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase.

A mammalian expression vector was designed to express a secreted soluble form of the UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase (polypeptide GalNAc transferase) with a metal binding site (HHWHHH) at the NH2 terminus. The recombinant enzyme was purified to homogeneity from COS-7 cell media by sequential chromatography on columns of NiCl2-chelating Sepharose, Affi-Gel blue, and Sephacryl S-100. Kinetic parameters of recombinant and native polypeptide GalNAc transferase were comparable for the donor UDP-GalNAc and for the peptide acceptor AcTPPP, EPO-T (PPDAATAAPLR), and HVF (PHMAQVTVGPGL). Initial velocity and product inhibition studies were carried out with purified recombinant polypeptide GalNAc transferase and the substrates UDP-GalNAc and peptide EPO-T. Initial velocity data was consistent with a sequential type mechanism in which binding of both substrates precedes product release. Product inhibition analysis using UDP showed competitive inhibition against UDP-GalNAc and a noncompetitive inhibition against peptide EPO-T. The dead end peptide analogue EPO-G (PPDAAGAAPLR) was a noncompetitive inhibitor of UDP-GalNAc and a competitive inhibitor of peptide EPO-T. Collectively, the results suggest that the most probable kinetic mechanism for the enzyme is one in which both substrates must bind in a random order prior to catalysis. Interestingly, the Km for EPO-T is similar to the Ki for EPO-G, suggesting that peptide interaction with the polypeptide GalNAc transferase does not require a hydroxyamino acid.

Mouth pressure in response to magnetic stimulation of the phrenic nerves.

BACKGROUND: Diaphragm strength can be assessed by the measurement of gastric (TW PGA), oesophageal (TW POES), and transdiaphragmatic (TW PDI) pressure in response to phrenic nerve stimulation. However, this requires the passage of two balloon catheters. A less invasive method of assessing diaphragm contractility during stimulation of the phrenic nerves would be of clinical value. A study was undertaken to determine whether pressure measured at the mouth (TW PM) during magnetic stimulation of the phrenic nerves accurately reflects TW POES, and to investigate the relations between TW PM and TW PDI; and also to see whether glottic closure and twitch potentiation can be avoided during these measurements. METHODS: Eight normal subjects and eight patients with suspected respiratory muscle weakness without lung disease were studied. To prevent glottic closure magnetic stimulation of the phrenic nerves was performed at functional residual capacity during a gentle expiratory effort against an occluded airway incorporating a small leak. TW PDI, TW POES, and TW PM were recorded. Care was taken to avoid potentiation of the diaphragm. RESULTS: In normal subjects mean TW PM was 13.7 cm H2O (range 11.3-16.1) and TW POES was 13.3 cm H2O (range 10.4-15.9) with a mean (SD) difference of 0.4 (0.81) cm H2O. In patients mean TW PM was 9.1 cm H2O (range 0.5-18.2) and TW POES was 9.3 (range 0.7-18.7) with a mean (SD) difference of -0.2 (0.84) cm H2O. The relation between TW PM and TW PDI was less close but was well described by a linear function. In patients with diaphragm weakness (low sniff PDI) TW PM was < 10 cm H2O. CONCLUSIONS: TW PM reliably reflects TW POES and can be used to predict TW PDI in normal subjects and patients without lung disease. TW PM may therefore be a promising non-invasive, non-volitional technique for the assessment of diaphragm strength.

Potentiation of diaphragmatic twitch after voluntary contraction in normal subjects.

BACKGROUND: Skeletal muscle twitch responses may be transiently increased by previous contractions, a phenomenon termed twitch potentiation. The aim of this study was to examine the extent and time course of diaphragmatic twitch potentiation and its relationship to both the magnitude and duration of the preceding voluntary diaphragmatic contraction. METHODS: Twitch transdiaphragmatic pressure (PDI) was measured in six normal subjects, before and after voluntary diaphragm contractions of 100%, 75%, 50%, and 25% of maximum PDI (PDImax) sustained for five and 10 seconds. RESULTS: Twitch PDI was significantly increased after 100%, 75%, and 50% contractions. Following maximal contractions sustained for 10 seconds the mean increase in twitch PDI was 52%. Following 50% contractions sustained for five seconds the mean increase in twitch height was 28%. In all runs twitch PDI returned to rested levels within 20 minutes. CONCLUSIONS: Twitch potentiation can be substantial, even following submaximal contractions, and must be taken into account when twitch pressure is used to assess diaphragm contractility.

Inspiratory muscle relaxation rate assessed from sniff nasal pressure.

BACKGROUND: Slowing of the maximum relaxation rate (MRR) of inspiratory muscles measured from oesophageal pressure (POES) during sniffs has been used as an index of the onset and recovery of respiratory muscle fatigue. The purpose of this study was to measure MRR at the nose (PNASAL MRR), to investigate its relationship with POES MRR, and to establish whether PNASAL MRR slows with respiratory loading. METHODS: Five normal subjects were studied. Each performed sniffs before and after two minutes of maximal isocapnic ventilation (MIV). In a separate session the subjects performed submaximal sniffs. POES and PNASAL were recorded during sniffs and the MRR (% pressure fall/10 ms) for each sniff was determined. RESULTS: Before MIV mean POES MRR was 8.9 and PNASAL MRR was 9.3. The mean (SD) difference between PNASAL MRR and POES MRR during a maximal sniff was 0.48 (0.34) (n = 64) and during submaximal sniffs was 0.28 (0.46) (n = 526). The subjects showed a mean decrease in sniff POES MRR of 27.4% (range 22.5-36%) after MIV and a similar reduction in sniff PNASAL MRR of 28.5% (range 24.1-41.3%). Both returned to control values within 5-10 minutes. CONCLUSIONS: PNASAL MRR reflects POES MRR over a wide range of sniff pressures, PNASAL MRR of maximal sniffs reflects POES MRR in normal subjects at rest and following MIV, so measurement of PNASAL MRR may be a useful non-invasive method for measuring inspiratory muscle MRR, thereby providing an index of respiratory muscle fatigue.

Comparison of cervical magnetic stimulation and bilateral percutaneous electrical stimulation of the phrenic nerves in normal subjects.

Cervical magnetic stimulation is a new technique for stimulating the phrenic nerves, and may offer an alternative to percutaneous electrical stimulation for assessing diaphragmatic strength in normal subjects and patients in whom electrical stimulation is technically difficult or poorly tolerated. We compared cervical magnetic stimulation with conventional supramaximal bilateral percutaneous electrical stimulation in nine normal subjects. We measured oesophageal pressure (Poes), gastric pressure (Pgas) and transdiaphragmatic pressure (Pdi). The maximal relaxation rate (MRR) was also measured. The mean magnetic twitch Pdi was 36.5 cmH2O (range 27-48 cmH2O), significantly larger than electrical twitch Pdi, mean 29.7 cmH2O (range 22-40 cmH2O). The difference in twitch Pdi was explained entirely by twitch Poes, and it is possible that the magnetic technique stimulates some of the nerves to the upper chest wall muscles as well as the phrenic nerves. We compared bilateral, rectified, integrated, diaphragm surface electromyographic (EMG) responses in three subjects and found results within 10% in each subject, indicating similar diaphragmatic activation. The within occasion coefficient of variation, i.e. same subject/same session, was 6.7% both for magnetic and electrical twitch Pdi. The between occasion coefficient of variation, i.e. same subject/different days, was 6.6% for magnetic stimulation and 8.8% for electrical. There was no difference between relaxation rates measured with either technique. We conclude that magnetic stimulation is a reproducible and acceptable technique for stimulating the phrenic nerves, and that it provides a potentially useful alternative to conventional electrical stimulation as a nonvolitional test of diaphragm strength.

Portable measurement of maximum mouth pressures.

We have compared a small portable mouth pressure meter (MPM) to our laboratory standard (LS) pressure recording equipment in order to evaluate this new device. The mouth pressure meter measures and displays as a digital read-out peak pressure for inspiratory and expiratory efforts. It samples the signal at 16 Hz, and an integral microprocessor is programmed to determine and display the maximum pressure averaged over one second both during inspiratory and expiratory manoeuvres (PImax and PEmax, respectively). A fine bore catheter connecting the mouthpiece of the mouth pressure meter to a Validyne pressure transducer enabled simultaneous measurement of pressure, which was analysed by LabVIEW, running on a Macintosh Quadra 700 computer. We studied 13 normal subjects and 11 patients with respiratory disease. Each subject performed inspiratory and five expiratory efforts. The values displayed from the mouth pressure meter were manually recorded. The mouth pressure meter reliably and accurately measured peak pressure and maximal pressure both for inspiratory and expiratory efforts in normals and patients. The mean +/- SD difference when compared with the Validyne method was 0.19 +/- 0.12 and -0.04 +/- 0.12 kPa, for PImax and PEmax, respectively. This portable device should be useful to measure mouth pressures, not only in the routine lung function laboratory but also at the bedside and in the clinic.