Robert Rauh

Influence of pneumoperitoneum and patient positioning on respiratory system compliance

STUDY OBJECTIVE To investigate the influence of pneumoperitoneum (PP) and posture on respiratory compliance and ventilation pressures. DESIGN Prospective, single blind trial. PATIENTS 10 female ASA physical status I and II patients scheduled for elective gynecologic laparoscopy. SETTING University medical center. INTERVENTIONS Anesthesia was performed as total IV anesthesia (TIVA) with propofol, alfentanil, and atracurium. After induction of anesthesia and orotracheal intubation, the lungs were ventilated to maintain partial pressure of CO(2) (P(ET)CO(2)) of 30 +/- 3 mmHg. Ventilation was kept constant. As gas mixture oxygen and air 1:1 was used without positive end-expiratory pressure (PEEP). MEASUREMENTS Measurements were taken before and after creation of pneumoperitoneum with an intraabdominal pressure (IAP) of 10 mmHg, of 15 mmHg in 20 degrees head-down tilt, then in 20 degrees head-up tilt, and after deflation of PP. We determined peak inspiratory pressure (PIP), mean airway pressure (mPaw), P(ET)CO(2), expiratory minute volume (V(E)), heart rate (HR), and systolic (SBP), diastolic (DBP), and mean arterial pressure (MAP). Respiratory system compliance (C(eff rs)) was calculated as quotient of tidal volume (V(T)) and PIP. MAIN RESULTS After creation of PP (IAP 10 mmHg), there was a significant increase of median PIP (3 cmH(2)O), mPaw (1 cm H(2)O) and arterial pressure (BP), (MAP by 7 mmHg), C(eff rs) decreased by 6 mL. cm H(2)O(-1). Increase of IAP to 15 mmHg led to a further increase of PIP (2 cm H(2)O) and mPaw (1 cm H(2)O), and a further decrease of C(eff rs) by 5 mL cm H(2)O(-1); BP decreased (MAP by 5.5 mmHg). Head-up or head down positions showed no significant hemodynamic or pulmonary changes. P(ET)CO(2)increased from 29.5 to 36 mmHg at an IAP of 15 mmHg, but then no further changes were noticed. Five minutes after deflation of pneumoperitoneum all values returned to baseline levels. CONCLUSIONS Creation of PP at an IAP of 15 mmHg reduced respiratory system compliance, and increased peak inspiratory and mean airway pressures, which quickly returned to normal values after deflation. Head-down or head-up position did not further alter those parameters.

Agreement of two different methods for measurement of heart rate variability

Abstract.Background: The widespread use of affordable devices with sufficient precision for measurement of heart rate variability (HRV) might lead to early detection of abnormalities in a large number of high-risk patients and athletes. The purpose of this study was to determine the limits of agreement of two devices for measuring HRV parameters differing in price and assumed precision. Subjects and methods: 36 healthy subjects (22 men and 14 women) with a mean age of 27.4 (SD 11.1) years were included. The two devices used for comparison were PowerLab® with Chart® software as the reference golden standard, and Polar® Transmitter®/Advantage® with Precision Performance® software, respectively. Measurements included the following heart rate variability parameters: heart rate, range of R-R-interval duration, SDNN, rMSSD, total Power, VLF power, LF power, and HF power. Measurements were taken during metronomic respiration over a total period of 3 minutes. Statistical analysis was performed according to Bland and Altman and by means of scatterplots and Spearman correlation coefficients. Results: Good agreement was found for heart rate (95 % CI of limits of agreement: −0.7–0.6 bpm; r = 0.999), range of duration of R-R-intervals (95 % CI: −18.9–17.0 ms; r = 0.997), rMSSD (95 % CI: −1.5–2.5 ms; r = 0.999), and SDNN (95 % CI: −3.0–3.1 ms; r = 0.997). Correlation of measurements was high for the variables total Power, VLF power, LF power, and HF power. Analysis of method agreement for frequency domain variables was statistically not feasible. Conclusion: The level of agreement for the analyzed time domain variables between the reference golden standard and the inexpensive device is sufficient to permit initial screening by family doctors, and self-administration by high-risk patients and athletes.

Mutations in the Amiloride-Sensitive Epithelial Sodium Channel in Patients With Cystic Fibrosis-Like Disease

We investigated whether mutations in the genes that code for the different subunits of the amiloride‐sensitive epithelial sodium channel (ENaC) might result in cystic fibrosis (CF)‐like disease. In a small fraction of the patients, the disease could be potentially explained by an ENaC mutation by a Mendelian mechanism, such as p.V114I and p.F61L in SCNN1A. More importantly, a more than three‐fold significant increase in incidence of several rare ENaC polymorphisms was found in the patient group (30% vs. 9% in controls), indicating an involvement of ENaC in some patients by a polygenetic mechanism. Specifically, a significantly higher number of patients carried c.–55+5G>C or p.W493R in SCNN1A in the heterozygous state, with odds ratios (ORs) of 13.5 and 2.7, respectively.The p.W493R‐SCNN1A polymorphism was even found to result in a four‐fold more active ENaC channel when heterologously expressed in Xenopus laevis oocytes. About 1 in 975 individuals in the general population will be heterozygous for the hyperactive p.W493R‐SCNN1A mutation and a cystic fibrosis transmembrane conductance regulator (CFTR) gene that results in very low amounts (0–10%) functional CFTR. These ENaC/CFTR genotypes may play a hitherto unrecognized role in lung diseases. Hum Mutat 30:1–11, 2009.

A mutation of the epithelial sodium channel associated with atypical cystic fibrosis increases channel open probability and reduces Na+ self inhibition

Increased activity of the epithelial sodium channel (ENaC) in the respiratory airways contributes to the pathophysiology of cystic fibrosis (CF), a genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. In some patients suffering from atypical CF a mutation can be identified in only one CFTR allele. We recently identified in this group of CF patients a heterozygous mutation (W493R) in the α‐subunit of ENaC. Here, we investigate the functional effects of this mutation by expressing wild‐type αβγENaC or mutant αW493RβγENaC in Xenopus oocytes. The αW493R mutation stimulated amiloride‐sensitive whole‐cell currents (ΔIami) by ∼4‐fold without altering the single‐channel conductance or surface expression of ENaC. As these data suggest that the open probability (Po) of the mutant channel is increased, we investigated the proteolytic activation of ENaC by chymotrypsin. Single‐channel recordings revealed that chymotrypsin activated near‐silent channels in outside‐out membrane patches from oocytes expressing wild‐type ENaC, but not in membrane patches from oocytes expressing the mutant channel. In addition, the αW493R mutation abolished Na+ self inhibition of ENaC, which might also contribute to its gain‐of‐function effects. We conclude that the αW493R mutation promotes constitutive activation of ENaC by reducing the inhibitory effect of extracellular Na+ and decreasing the pool of near‐silent channels. The resulting gain‐of‐function phenotype of the mutant channel might contribute to the pathophysiology of CF in patients carrying this mutation.

Influence of pneumoperitoneum and patient positioning on preload and splanchnic blood volume in laparoscopic surgery of the lower abdomen

STUDY OBJECTIVE To determine the hemodynamic effects of pneumoperitoneum and patient positioning during laparoscopic surgery of the lower abdomen. DESIGN Prospective study. SETTING University-affiliated medical center. PATIENTS 10 ASA physical I and II female patients scheduled for laparoscopic surgery of the lower abdomen. INTERVENTIONS Patients were anesthetized with propofol and an alfentanil infusion, then intubated, and normoventilated. MEASUREMENTS After intubation, a transesophageal multiplane probe for measurements of right (RVESA) and left (LVESA) ventricular end-systolic and end-diastolic areas (RVEDA and LVEDA) and ejection fraction area (RVEFa, LVEFa) was introduced; heart rate (HR) and noninvasive blood pressure (BP) were recorded every minute. Ventilation was not changed during the measurements. A transvaginal ultrasound probe was inserted to measure the diameter of the common iliac vein. Measurements were performed 15 minutes after induction of anesthesia and while patients were in the supine position (P 0), 10 minutes after CO(2) insufflation to 10 mmHg IA pressure (P 10), 10 minutes after a further increase to 15 mmHg (P 15), 10 minutes after 20 degrees Trendelenburg (P 15 T), and 20 degrees reverse Trendelenburg positions (P 15 RT). Data are shown as medians, 25th to 75th percentiles, and comparisons between P 0, P 10, P 15, and P15 T were made with the Friedman test, followed by Wilcoxon test, when significant. Data at P 15 T, P 15 RT, and P 15 were compared using the Wilcoxon test, with a p-value < 0.05 regarded as significant. MAIN RESULTS Pneumoperitoneum at 10 mmHg abdominal pressure caused a significant increase of LVESA by 78% (RVESA: 61%) and LVEDA by 48.5% (RVEDA: 45%). The diameter of the common iliac vein was decreased by 6%. A further increase of abdominal pressure to 15 mmHg led to an additional increase of 20% (LVESA) and 17% (LVEDA). Mean arterial pressure increased by a significant 7% at P 10, decreasing subsequently by 5% at P 15. The Trendelenburg position did not alter any hemodynamic findings. Reverse Trendelenburg position, however, caused a significant LVEDA-and RVEDA-decrease by 18% and 27%, respectively, and an increase in the diameter of the common iliac vein by 22%. The LVEFa and RVEFa decreased significantly after abdominal CO(2) insufflation by 18% each (P 10) without further change. CONCLUSIONS The lithotomy position and subsequent pneumoperitoneum increased preload, probably as a result of blood shifting from the abdomen to the thorax by compression of splanchnic vessels caused by the pneumoperitoneum. Careful fluid management, maintaining low abdominal pressure, and use of the reverse Trendelenburg position are favored to prevent adverse hemodynamic effects in laparoscopic surgery.

The δ-Subunit of the Epithelial Sodium Channel (ENaC) Enhances Channel Activity and Alters Proteolytic ENaC Activation

The epithelial sodium channel (ENaC) is probably a heterotrimer with three well characterized subunits (αβγ). In humans an additional δ-subunit (δ-hENaC) exists but little is known about its function. Using the Xenopus laevis oocyte expression system, we compared the functional properties of αβγ- and δβγ-hENaC and investigated whether δβγ-hENaC can be proteolytically activated. The amiloride-sensitive ENaC whole-cell current (ΔIami) was about 11-fold larger in oocytes expressing δβγ-hENaC than in oocytes expressing αβγ-hENaC. The 2-fold larger single-channel Na+ conductance of δβγ-hENaC cannot explain this difference. Using a chemiluminescence assay, we demonstrated that an increased channel surface expression is also not the cause. Thus, overall channel activity of δβγ-hENaC must be higher than that of αβγ-hENaC. Experiments exploiting the properties of the known βS520C mutant ENaC confirmed this conclusion. Moreover, chymotrypsin had a reduced stimulatory effect on δβγ-hENaC whole-cell currents compared with its effect on αβγ-hENaC whole-cell currents (2-fold versus 5-fold). This suggests that the cell surface pool of so-called near-silent channels that can be proteolytically activated is smaller for δβγ-hENaC than for αβγ-hENaC. Proteolytic activation of δβγ-hENaC was associated with the appearance of a δ-hENaC cleavage product at the cell surface. Finally, we demonstrated that a short inhibitory 13-mer peptide corresponding to a region of the extracellular loop of human α-ENaC inhibited ΔIami in oocytes expressing αβγ-hENaC but not in those expressing δβγ-hENaC. We conclude that the δ-subunit of ENaC alters proteolytic channel activation and enhances base-line channel activity.

Cholesterol Depletion of the Plasma Membrane Prevents Activation of the Epithelial Sodium Channel (ENaC) by SGK1

The lipid environment of the epithelial sodium channel (ENaC) and its possible association with so-called lipid rafts may be relevant to its function. The aim of our study was to confirm the association of ENaC with lipid rafts and to analyze the effect of cholesterol depletion of the plasma membrane by methyl-β-cyclodextrin (MβCD) on channel function and regulation. Using sucrose density gradient centrifugation we demonstrated that a significant portion of ENaC protein distributes to low density fractions thought to be typical lipid raft fractions. Importantly, cholesterol depletion of cell lysate by MβCD shifted ENaC to non-raft fractions of higher density. Live cell imaging demonstrated that treatment with MβCD largely reduced filipin staining over time, confirming cholesterol depletion of the plasma membrane. For electrophysiological studies intact oocytes were exposed to 20 mM MβCD for three hours. MβCD treatment had no consistent effect on baseline whole-cell ENaC currents. In addition to the typical single channel conductance of about 5 pS, subconductance states of ENaC were occasionally observed in patches from MβCD treated but not from control oocytes. Importantly, in outside-out patch clamp recordings the stimulatory effect of recombinant SGK1 in the pipette solution was essentially abolished in oocytes pretreated with MβCD. These results indicate that ENaC activation by cytosolic SGK1 is compromised by removing cholesterol from the plasma membrane. Thus, ENaC activation by SGK1 may require the presence of an intact lipid environment and/or of lipid rafts as signalling platform.

Acute effects of caffeine on heart rate variability in habitual caffeine consumers.

During the last years, heart rate variability (HRV) has become a promising risk factor for cardiovascular events. However, the effect of caffeine on HRV in habitual caffeine consumers has barely been investigated. Therefore, we treated 30 male habitual caffeine users in a randomized double‐blinded crossover study design with either placebo, 100 or 200 mg caffeine orally and determined parameters of HRV under resting conditions and metronomic breathing. As result, we could not detect significant differences in HRV parameters up to 90 min after drug ingestion. We conclude that modest amounts of caffeine do not reveal negative nor positive effects on HRV within the first 90 min after drug ingestion in young and healthy habitual caffeine consumers. However, further research is necessary to determine the effects of caffeine on HRV in habitual caffeine users, healthy as well as suffering from diabetes, hypertension and postmyocardial infarction.

(NDRG2) Stimulates Amiloride-sensitive Na+ Currents in Xenopus laevis Oocytes and Fisher Rat Thyroid Cells

Regulation of the epithelial sodium channel (ENaC) is highly complex and may involve several aldosterone-induced regulatory proteins. The N-Myc downstream-regulated gene 2 (NDRG2) has been identified as an early aldosterone-induced gene. Therefore, we hypothesized that NDRG2 may affect ENaC function. To test this hypothesis we measured the amiloride-sensitive (2 μm) whole cell current (ΔIami) in Xenopus laevis oocytes expressing ENaC alone or co-expressing ENaC and NDRG2. Co-expression of NDRG2 significantly increased ΔIami in some, but not, all batches of oocytes tested. An inhibitory effect of NDRG2 was never observed. Using a chemiluminescence assay we demonstrated that the NDRG2-induced increase in ENaC currents was accompanied by a similar increase in channel surface expression. The stimulatory effect of NDRG2 was preserved in oocytes maintained in a low sodium bath solution to prevent sodium feedback inhibition. These findings suggest that the stimulatory effect of NDRG2 is independent of sodium feedback regulation. Furthermore, the stimulatory effect of NDRG2 on ENaC was at least in part additive to that of Sgk1. A short isoform of NDRG2 also stimulated ΔIami. Overexpression of NDRG2 and ENaC in Fisher rat thyroid cells confirmed the stimulatory effect of NDRG2 on ENaC-mediated short-circuit current (ISC-ami). In addition, small interference RNA against NDRG2 largely reduced ISC-ami in Fisher rat thyroid cells. Our results indicate that NDRG2 is a likely candidate to contribute to aldosterone-mediated ENaC regulation.

Stimulation of the epithelial sodium channel (ENaC) by the serum- and glucocorticoid-inducible kinase (Sgk) involves the PY motifs of the channel but is independent of sodium feedback inhibition

The epithelial sodium channel (ENaC) is the major mediator of sodium transport across the apical membranes of the distal nephron, the distal colon, the respiratory tract and the ducts of exocrine glands. It is subject to feedback inhibition by increased intracellular Na+, a regulatory system wherein the ubiquitin protein ligases, Nedd4 and Nedd4-2, bind to conserved PY motifs in the C-termini of ENaC and inactivate the channel. It has been proposed recently that the kinase Sgk activates the channel as a consequence of phosphorylating Nedd4-2, thus preventing it from inhibiting the channels. This proposal predicts that Sgk should interfere with Na+ feedback regulation of ENaC. We have tested this prediction in Xenopus laevis oocytes and in mouse salivary duct cells and found that in neither system did increased activity of Sgk interrupt Na+ feedback inhibition of ENaC. We found, however, that Sgk stimulation was largely abolished in oocytes expressing ENaC channels with C-terminal truncations or mutated PY motifs. We were also unable to confirm that Sgk directly interacts with Nedd4-2 in vitro. We conclude that the stimulatory effect of Sgk on ENaC requires the presence of the channel’s PY motifs, but it is not due to the interruption of Na+ feedback regulation.