Serum carbonic anhydrase III,an enzyme of type I muscle fibres,and the intensity of physical exercise

The effects of 30 min running with stepwise increasing intensity (exhaustive, energy demand approx. 50 100% ofVO_2max), 60 s supramaximal running (anaerobic, 125% ofVO_2max) and 40–60 min low-intensity running (acrobic, 40–60% ofVO_2max) on serum concentration of muscle-derived proteins were studied in 5 male and 5 female elite orienteerers. S-Carbonic anhydrase III (S-CA III) was used as a marker of protein leakage from type I (slow oxidative) muscle fibres and S-myoglobin (S-Mb) as a non-selective (type I+II) muscular marker. The fractional increase in S-CA III (S-Ca III) was 0.37±0.09 (mean±SEM,p<0.001), 0.10±0.05 (N. S.) and 0.46±0.09 (p<0.001) 1 h after exhaustive, anaerobic and aerobic exercise, respectively. The corresponding values for S-Mb were 1.45±0.36 (p<0.001), 0.39±0.13 (p<0.01) and 0.67±0.18 (p<0.001). The value for the S-CA III/S-Mb ratio was 0.68±0.03 after the acrobic exercise, but only 0.25–0.26 (p vs. aerobic exercise <0.001) after the two high-intensity forms of exercise. Since type I fibres of skeletal muscle are known to be responsible for power production during low-intensity exercise, whereas fibres of both type I and type II are active at higher intensities, the S-CA III/S-Mb ratio may depend on the recruitment profile of type I vs. type I+II fibres.     

Time-dependent effect of sectioning carotid sinus nerves on the vagally mediated baroreflex

The purpose of the reported experiments was to examine whether the vagally mediated baroreflex system (V-system) increases its feedback gain with time after sectioning of the carotid sinus nerves (CSN). In 10 dogs anesthetized with Nembutal, we determined the overall open-loop gain (G) of the rapidly acting arterial pressure control system. G was assessed as (AP_I/AP_S)-1, where AP_I and AP_S represent the immediate and steady-state decreases in arterial pressure at the aortic arch following a fast step-wise reduction in blood volume. AP_I, AP_S and G_INTACT in the intact condition (30th min before sectioning of the CSN) were –1.8±0.1 kPa, –0.20±0.01 kPa and 8.4±0.3 (mean±SE), respectively. The mean values of AP_I, AP_S and G after sectioning of the CSN (G_V), first averaged within individual dogs and then averaged for ten dogs, were –2.53±0.07 kPa, –1.1±0.05 kPa, and 1.5±0.1, respectively. G_V did not change with time over about 4 h after sectioning of the CSN. It is concluded that the V-system cannot augment its ability to restore arterial pressure in compensation for the lost function of the carotid sinus baroreflex system over 4 h after sectioning of the CSN in the anesthetized dog.     

Thermal characterization and transmitter analysis of single units in the preoptic and anterior hypothalamus of conscious ducks

With a multibarrel assembly combining one carbon fiber micropipette as recording electrode and 6 pipettes for microiontophoretic application of drugs, the activity of neurons in the preoptic and anterior hypothalamic (POAH) region was extracellularly recorded in situ in conscious ducks implanted chronically with a device permitting hypothalamic thermal stimulation. Among 355 neurons 17% were identified as warm-responsive (warm units) and 20% as cold-responsive (cold units). In 58 warm and 56 cold units control discharge rates at 40°C local temperature (F₄₀) and temperature coefficients (F/T) were determined and presented as means ± SEM. The F₄₀ values of warm units (35.2±2.3 Imp · s^–1) were significantly higher than of cold units (16.3±1.8 Imp · s^–1). The F/T values (+1.77±0.15 and –1.77±0.19 Imp · s^–1 · °C^–1) of warm and cold units were not different in absolute terms. In pilot experiments either activation or inhibition by lowering whole-body temperature was observed in both warm and cold units. Microiontophoretic application of one or more of the amines acetylcholine (ACh), 5-hydroxytryptamine (5-HT), and noradrenaline (NA) to warm and cold units revealed differences in their responsiveness to ACh, which more consistently stimulated cold units. NA inhibited the majority of warm units; 5-HT stimulated the majority of cold units. In both warm and cold units NA and ACh differed in their actions, with the latter amine more consistently producing activation.     

An electrophysiological study of angiotensin II regulation of Na-HCO3 cotransport and K conductance in renal proximal tubules

The effect of picomolar concentrations of angiotensin II (AII) was investigated in isolated perfused rabbit renal proximal tubules using conventional or pH-sensitive intracellular microelectrodes. Under control conditions cell membrane potential (V _b) and cell pH (pH_i) averaged –53.8±1.9 mV (mean±SEM,n=49) and 7.24±0.01 (n=10), respectively. AII (at 10^–11 mol/l), when applied from the bath (but not when applied from the lumen perfusate), produced the following effects: approximately 85% of the viable tubules responded with a small depolarization (+ 5.5±0.4 mV,n=43) which was accompanied in half of the pH_i measurements by a slow acidification (pH_i=–0.03±0.01,n=5). The remaining 15% responded with a small hyperpolarization (V_b=–3.1±0.4 mV,n=6). All changes were fully reversible and repeatable. Experiments with fast changes in bath HCO₃ or K concentrations, as well as measurements of the basolateral voltage divider fraction in response to transepithelial current flow, explain these observations as stimulation of a basolateral Na-HCO₃ cotransporter and of a basolateral K conductance. Both counteract in their effect onV _b, but can be individuated by blocker experiments with 4,4-diisothiocyanatostilbene-2,2-disulphonic acid (DIDS) and barium. Both the stimulation of Na-HCO₃ cotransport and the stimulation of the K conductance may result from down-regulation of the level of cyclic adenosine monophosphate in the cell.     

Volume flow,hydraulic conductivity and electrical properties across bovine tracheal epithelium in vitro: Effect of histamine

Volume flow (J _v), potential difference (), shortcircuit current (i ₀) and electrical resistance (R) were measured simultaneously across bovine tracheal epithelium in vitro. Under basal conditions, with no applied hydrostatic or osmotic pressure gradient (P=0, =0), no spontaneousJ _v was observed. was 31±2 mV (lumen negative),i ₀ 161±8 A cm^–2 andR 202±9 cm²,n=50. When a was applied, by adding 20–80 mM sucrose into the medium bathing either the luminal or the serosal side of the tissue, a linear relationship was found between andJ _v toward the lumen or toward the serosa. The apparent hydraulic conductivity (apparentL _p) was 4.6–4.910^–6 cm s^–1 atm^–1. Histamine 10^–4 M did not induce any spontaneousJ _v under basal conditions and had no effect oni ₀ nor onR. However, histamine caused a 100% increase inJ _v elicited by sucrose gradients. It was concluded that histamine exerts a selective action on the hydraulic conductivity of bovine tracheal epithelium. Experiments using H₁-receptors antagonists (diphenhydramine, dimetindene, chloropyramine) and H₂-antagonists (cimetidine, metiamide) or a H₂-agonist (impromidine) showed that the increase ofL _p induced by histamine was mediated via H₂-receptors.Supported by the Swiss National Foundation (SNF), grant no. 3.5880.79     

Electrically evoked isometric and isokinetic properties of the triceps surae in young male subjects

Summary The relationship between electrically evoked isometric and isokinetic properties of the triceps surae have been studied in 11 healthy male subjects. The results showed that the time to peak tension (TPT) and half relaxation time (1/2 RT) of the maximal twitch were 110±11 ms and 82±11 ms respectively, and the peak rates of rise of contraction (P ₅₀, P ₂₀₀) and relaxation (P _R50, P _R200) at 50 and 200 Hz were 0.36±0.07, 0.48±0.08 and 1.27±0.33, 1.25±0.27% Po ms^–1 respectively. The decline in force during a fatigue test was significantly (P<0.02) associated with the decrease in maximal relaxation rate (r=0.79). The TPT was significantly (P<0.05) and inversely related to P ₅₀ and P ₂₀₀. The mean angle specific torque-velocity relationship for the 11 subjects was adequately described by the empirical exponential equation of the form: V=16.5 (e ^–p/30.8–e ^–84.3/30.8) where V=velocity (rads s^–1) P=torque (Nm). The only significant association found between the isometric and isokinetic properties of the muscle was between P _R200 and the torque expressed at a given velocity of 4 rads s^–1. This lack of association between the two variables is difficult to explain with certainty but it is suggested that it may be due to the differential effects of Ca²⁺ release and uptake and cross-bridge turnover rate in the two situations.     

Effects of fatigue and altered pH on isometric force and velocity of shortening at zero load in frog muscle fibres

Summary Single skeletal muscle fibres ofR. temporaria (0.9–2.5° C) were stimulated to produce a 1 s isometric tetanus at regular intervals until constant mechanical responses were attained. Various degrees of force depression (fatigue) were produced by decreasing the contraction interval from 30 or 15 min (control) to 120, 60, 30 and 15 s, respectively. In this way the steady-state tetanic force could be reversibly reduced to approximately 70% of the control value. The velocity of shortening at zero load,V ₀, was determined at each level of fatigue using an approach for direct measurement ofV ₀. V ₀ was not significantly affected as long as the decrease in force was less than 10%. With further reduction of the isometric tension there was a progressive decline ofV ₀ according to the following empirical relationship between percentage depression of force (P ₀) and maximum speed (V ₀) of shortening: V ₀=0.006 P ₀ ^2.48 –1.0 (correlation coefficient, 0.86). Cine photographic recording of nylon markers on the fibre surface provided evidence that fatigue developed uniformly along the fibre with no sign of failure of excitation in any segment.The change in mechanical performance during fatigue could be reproduced in the non-fatigued fibre by reducing the pH of the external medium within the range 8.0–6.6 using a bicarbonate-CO₂ buffer. A decrease in pH thus reduced both the rate of rise and the total amplitude of isometric force and prolonged the relaxation phase. Furthermore, there was a drop inV ₀ that was related to the force decline in approximately the same way as observed during fatigue.The results support the idea that fatigue involves both a reduced state of activation of the contractile system and a specific (activation independent) inhibition of crossbridge turnover. Increased intracellular H⁺ concentration is likely to contribute to the development of both these effects during fatigue.     

Separating the direct effect of hypoxia from the indirect effect of changes in cardiac output on the maximum pressure difference across the tricuspid valve in healthy humans

In healthy humans, changes in cardiac output are commonly accommodated with minimal change in pulmonary artery pressure. Conversely, exposure to hypoxia is associated with substantial increases in pulmonary artery pressure. In this study we used non-invasive measurement of an index of pulmonary artery pressure, the maximum systolic pressure difference across the tricuspid valve (P_max), to examine the pulmonary vascular response to changes in blood flow during both air breathing and hypoxia. We used Doppler echocardiography in 33 resting healthy humans breathing air over 6–24 h to measure spontaneous diurnal variations in P_max and cardiac output. Cardiac output varied by up to ~2.5 l/min; P_max varied little with cardiac output [0.61±0.74 (SD) mmHg min l^–1]. Eight of the volunteers were also exposed to eucapnic hypoxia (end-tidal ) for 8 h. In this group P_max rose progressively from 21 mmHg to 37 mmHg over 8 h. By comparing diurnal variations in P_max during air breathing with changes in P_max during hypoxia in the same eight individuals, we concluded that only approximately 5% of the changes in P_max during hypoxia could be attributed to concurrent changes in cardiac output. The low sensitivity of P_max to changes in cardiac output makes it a useful index of hypoxic pulmonary vasoconstriction in healthy humans.     

Plasma catecholamines and heart rate at the beginning of muscular exercise in man

Summary The relationship between the time course of heart rate and venous blood norepinephrine (NE) and epinephrine (E) concentrations was studied in 7 sedentary young men before and during 3 bicycle exercises of 5 min each (respectively 23±2.8%, 45±2.6% and 65±2.4% , mean ±SE). During the low level exercise the change in heart rate is monoexponential ( =5.7±1.2s) and no increment above the resting level of NE (NE) or of E (E) occurs. At the medium and highest intensity of exercise: a) the change in heart rate is biexponential, for the fast and the slow component averaging about 3 and 80 s respectively; b) NE (but not E) increases continuously with time of exercise; c) at the 5th min of exercise heart rate increments are related to NE; d) between 20s and 5 min, at corresponding sampling times, the heart rate of the slow component is linearly related to NE. At exercise levels higher than 33% the increase in heart rate described by the slow component of the biexponential kinetic could be due to an augmented sympathetic activity revealed by increased NE blood levels.     

The slow component of oxygen uptake during intense, sub-maximal exercise in man is associated with additional fibre recruitment

Single muscle fibre metabolites and pulmonary oxygen uptake (O₂) were measured during moderate and intense, sub-maximal exercise to test the hypothesis that additional fibre recruitment is associated with the slow component of O₂. Seven healthy, male subjects performed 20 min moderate (MOD, ~50% of O_2,max) and intense (INT, ~80% O_2,max) cycling at 70 rpm. Glycogen content decreased significantly in type I and IIa fibres during INT, but only in type I fibres during MOD. During INT, creatine phosphate (CP) content decreased significantly both in types I and II fibres in the first 3 min (CP: 16.0±2.7 and 16.8±4.7 mmol kg^–1 d.w., respectively) and in the next 3 min (CP: 16.2±4.9 and 25.7±6.7 mmol kg^–1 d.w., respectively) with no further change from 6–20 min. CP content was below the pre-exercise level (mean–1 SD) in 11, 37, 70 and 74% of the type I fibres after 0, 3, 6 and 20 min of INT, respectively, and in 13, 45, 83 and 74% of the type II fibres. During INT, O₂ increased significantly by 6±1 and 4±1% in the periods 3–6 and 6–20 min, respectively (O_2,(6–3min): 0.14±0.02 l min^–1), whereas O₂ was unchanged from 3 to 20 min of MOD. Exponential fitting revealed a slow component of O₂ during INT that appeared after ~2.6 min and amounted to 0.24 l min^–1. The present study demonstrates that additional type I and II fibres are recruited with time during intense sub-maximal exercise in temporal association with a significant slow component of O₂.     

Effect of primary,secondary and tertiary amines on membrane potential and intracellular pH in Xenopus laevis oocytes

The effects of primary, secondary and tertiary methyl- and ethylamines as well as of quaternary ammonium compounds on membrane potential, V _m, and intracellular pH (pH_i) of oocytes from Xenopus laevis were studied using electrophysiological methods. The quaternary ammonium compounds, tetramethyl- (TMA) and tetraethyl- (TEA) ammonium chloride and choline chloride (each 10 mmol/l), affected V _m only slightly. In contrast, primary, secondary and tertiary amines strongly depolarized V _m. Depolarization was inversely proportional to the pK _a of the amines. Trimethylamine (pK _a 9.8) depolarized V _m by 61.7±21.8 mV (n=13) and exerted its half-maximal effect at less than 2 mmol/l. In paired experiments (n=6), trimethylamine (10 mmol/l) reduced V _m only by 5.1±1.3 mV at a bath pH of 6.0, but by 73.2±20.0 mV at pH 7.5, suggesting that the deprotonated, uncharged form of the amines was responsible for the depolarization. pH_i measurements using the Fluka pH-sensitive cocktail 95 293 revealed a short initial alkalinization and a subsequent acidification in the presence of trimethylamine (10 mmol/l). The intracellular acidification proceeded much more slowly than the depolarization. As shown by measurements using a two-electrode voltage-clamp device, the depolarization was associated with an inward current. This trimethylamine-sensitive current, I _m, decreased from-128±82 nA (n=4) at a clamp potential V _c=-70 mV to-3±33 nA at V _c=0 mV. Neither V _m nor I _m were markedly inhibited by GdCl₃, BaCl₂, or amiloride (each 1 mmol/l). Only 1 mmol/l diphenylamine-2-carboxylate (DPC) diminished both responses. The data suggest that the amines modify anion or cation conductances of the oocytes by as yet unknown mechanisms.     

Relationship between muscle architectural features and oxygenation status determined by near infrared device

This study tested whether regional differences in oxygenation status could result from differences in muscle fiber architecture. Architectural properties, oxygen supply, and consumption in the medial head of the gastrocnemius muscle (GM) were determined in vivo in six men using B-mode ultrasound and functional near infrared (NIR) imaging devices. Fascicle length, fascicle angle, NIR-O₂ saturation (deoxygenated Hb or oxygenated Hb), and NIR-blood volume (sum of deoxygenated and oxygenated Hb) were obtained in the distal and proximal portions of the GM at rest and during contraction. Exercise consisted of 2 min of standing plantar flexion at 1 Hz with an additional load of 50% of each subjects weight. Plantar flexion produced larger decreases (: difference between rest and exercise values) in NIR-O₂ saturation [mean saturation (SD) of 0.14 (0.05) vs 0.07 (0.04) optical density units] and NIR-blood volume [mean saturation (SD) of –0.23 (0.08) vs –0.13 (0.04) optical density units] in the distal compared with the proximal portion (P<0.05 for all comparisons). It also produced larger changes () in fascicle length [mean length (SD) of –16.5 (4.7) vs –8.2 (4.2) mm] and fascicle angle (mean angle (SD) of 10.8 (1.4)° vs 3.9 (2.1)°] in the distal compared with the proximal portion (P<0.05 for all comparisons). There were significant correlations between NIR-O₂ saturation and fascicle length (r=–0.84, P<0.05), and between NIR-O₂ saturation and fascicle angle (r=–0.90, P<0.05), between NIR-blood volume and fascicle length (r=0.91, P<0.05), between NIR-blood volume and fascicle angle (r=–0.85, P<0.05). In conclusion, the plantar flexion exercise produced regional differences in oxygenation status consistent with regional differences in muscle architecture.     

Influence of glucose absorption on ion activities in cells and submucosal space in goldfish intestine

Mucosal glucose addition evokes in goldfish intestinal epithelium a fast depolarization of the mucosal membrane potential (_mc = 12 mV) followed by a slower repolarization (_mc = –7 mV). The intracellular sodium activity, a_iNa⁺, rises from 13.2±2.4 meq/l by 6.7±0.5 meq/l within 5 min, a_iCl^– rises about 3 meq/l above the control value of 37.7±2.2 meq/l, while a_iK is constant (97.7 ±7.4 meq/l). The potassium activity measured in the submucosal interstitium near the basal side of the cells (a_iK⁺) is 5.2±0.2 meq/l in non-absorbing tissue compared to 4.2 meq/l in the bathing solution and shows a transient increase due to glucose absorption (1.1±0.1 meq/l).In chloride-free media a_sK⁺=4.2±0.1 meq/l and _mc hyperpolarizes by –13 mV. The depolarization due to glucose absorption increases (_mc = 14.1 ± 1.4 mV) and the repolarization ( _mc ^repol ) disappears. In addition, a_iNa⁺ rises from 16.3±2.4 meq/l by 9.9±1.5 meq/l within 5 min, a_iK⁺ remains constant and equal to the value in chloride containing solutions (88.5±2.8 meq/l); a_sK⁺ increases transiently (1.1±0.1 meq/l).Serosal Ba²⁺ (5 mM) depolarizes _mc (+14.2±1.0 mV) and abolishes the repolarization. Increased serosal or mucosal potassium activity depolarizes _mc and abolishes the repolarization.These effects are discussed in terms of changes of ion activities, the basolateral potassium conductance, the influence of intracellular Ca²⁺, the functional state of the Na/K-pump, and modulation of membrane permeabilities by extracellular potassium.     

Axial heterogeneity of sodium-bicarbonate cotransport in proximal straight tubule of rabbit kidney

Intracellular microelectrodes were used to investigate rheogenic Na⁺(HCO ₃ ^– ) _n cotransport in different segments of isolated proximal straight tubule (PST) of rabbit kidney. In the first portion (S2 segment) the peritubular cell membrane potentialV _b averaged –46.0, SE±1.3 mV (n=20), while in the terminal portion (S3 segment) it averaged –68.3, SE±2.5 mV (n=10). This difference may reflect different modes of anion permeation across the peritubular cell membrane. In S2 segments, sudden 101 reduction of bath HCO ₃ ^– concentration caused a fast transient cell depolarization, V _b=–45.8, SE±1.2 mV (n=33) as expected from the presence of Na⁺(HCO ₃ ^– ) _n contransport. As the puncture site moved further distally, V _b declined and gradually changed its time course by superposition of a slower secondary depolarization. In this region the transient cell depolarization could be recuperated by inhibiting the peritubular K⁺ conductance with Ba²⁺ (1 mmol/l). In S3 segments, however, the HCO ₃ ^– -dependent transient cell depolarization was completely lost both in the absence and presence of Ba²⁺. In addition, sudden reduction of bath Na⁺ concentration did not acidify the cell, as it did in the S2 segment. The data indicate that the expression of Na⁺(HCO ₃ ^– ) _n cotransport in the peritubular cell membrane gradually diminishes towards the end of the S2 segment and is lost in the S3 segment.     

Effect of menstrual cycle and gender on ventilatory and heart rate responses at the onset of exercise

To clarify the luteal-follicular and male–female differences in ventilatory and heart rate responses at the onset of exercise, seven women and seven men performed voluntary exercise and passive movement for 20 s (brief voluntary exercise and brief passive movement) and voluntary exercise for 3 min (long voluntary exercise) in a sitting position. Voluntary exercise consisted of alternate flexion-extensions of both lower legs with a weight corresponding to about 2.5% of the subjects' body mass attached to each ankle, at a frequency of about 60 times min^–1. Passive movement was carried out without weights by experimenters pulling ropes attached to both of the subjects' ankles, in the same way as voluntary exercise. During these exercises and movements, minute inspiratory ventilation (_I) and heart rate (HR) were continuously measured by breath-by-breath and beat-to-beat techniques. We calculated relative changes of _I and HR (_I and HR). Additionally, we averaged _I and HR obtained during the exercise and movement for each subject, and performed a correlation analysis between the averaged _I and HR. It was clarified that: (1) _I and HR in the follicular phase were almost equal to those in the luteal phase; (2) there were no significant male–female differences in these parameters; (3) significant positive correlations were found in both genders only during brief voluntary exercise. We conclude that ventilatory and HR responses at the onset of voluntary exercise and passive movement are not affected by the menstrual cycle or gender.     

N-Acetyl-l-cysteine and its derivatives activate a Cl− conductance in epithelial cells

N-Acetyl-l-cysteine (NAC) is a widely used mucolytic drug in patients with a variety of respiratory disorders including cystic fibrosis (CF). The beneficial effects of NAC are empirical and the exact mechanism of action in the airways remains obscure. In the present study we examined the effects on whole-cell (we) conductance (G _m) and voltage (V _m) of NAC and the congeners S-carboxymethyl-l-cysteine (CMC) andS-carbamyl-L-cysteine (CAC) andL-cysteine in normal and CF airway epithelial cells.L-Cysteine (1 mmol/1) had no detectable effect. The increase inG _m (G_m) by the other compounds was concentration dependent and was (all substances at 1 mmol/1) 3.8 ± 1.4 nS (NAC; n = 11), 4.2 ± 1.0 nS (CMC;n = 16) and 3.8 ± 1.6 nS (CAC;n = 18), respectively. The changes in G_m were paralleled by an increased depolarization (V_m) when extracellular Cl^– concentration was reduced to 34 mmol/1: under control conditions = -4.1 ± 2.1 versus 10.2 ± 2.1 mV in the presence of NAC, CMC, CAC (n = 36). In the presence of NAC, CMC and CAC, the reduction in Cl^– concentration was paralleled by a reduction ofG _m by 2.1 ± 0.4 nS (n = 35), indicating that all substances acted by increasing the Cl^– conductance. Analysis of intracellular pH did not reveal any changes by any of the compounds (1 mmol/1). A Cl^– conductance was also activated in HT₂₉ colonic carcinoma and CF tracheal epithelial (CFDE) cells but not in CFPA1 cells, which do not express detectable levels of F508-CFTR, suggesting that the presence of CFTR may be a prerequisite for the induction of Cl^– currents. Next we examined the ion currents in Xenopus oocytes microinjected with CFTR-cRNA. Water-injected oocytes did not respond to activation by forskolin and 3-isobutyl-l-methylxanthine (IBMX) (Gm = 0.08 ±0.04 S;n = 10) and no current was activated when these oocytes were exposed to NAC or CMC. In contrast, in CFTR-cRNA-injected cocytesG _m was enhanced when intracellular adenosine 3,5-cyclic monophosphate (cAMP) was increased by forskolin and IBMX (G _m = 4.5 ± 1.3 S;n = 8).G _m was significantly increased by 0.74 ± 0.2 S (n = 11) and 0.46 ± 0.1 S (n = 10) when oocytes were exposed to NAC and CMC, respectively (both I mmol/1). In conclusion, NAC and its congeners activate Cl^– conductances in normal and CF airway epithelial cells and hence induce electrolyte secretion which may be beneficial in CF patients. CFTR appears to be required for this response in an as yet unknown fashion.     

Amino acids 519–524 of Dictyostelium myosin II form a surface loop that aids actin binding by facilitating a conformational change

Residues 519–524 of Dictyostelium myosin II form a small surface loop on the actin binding face, and have been suggested to bind directly to actin through high affinity hydrophobic interactions. To test this hypothesis, we have characterized mutant myosins that lack this loop in vivo and in vitro. A mutant myosin in which this loop was replaced by an Ala residue (519–524/+A) was non-functional in vivo. Replacement with a single Gly residue instead of Ala yielded partial function, suggesting that structural flexibility, rather than hydrophobicity, is the key feature of the loop. The in vivo phenotype of the mutant enabled us to identify a number of additional amino acid changes that restore function to the 519–524/+A mutation. Intriguingly, many of these, including L596S, were located at some distances away from the 519–524 loop. We have also isolated suppressors for the L596S mutant myosin, which was not functional in vivo. The suppressors for 519–524/+A and those for L596S showed complementary charge patterns. In ATPase assays, 519–524/+A S1 showed very low activity and little enhancement by actin, whereas L596S S1 was hyper active and displayed enhanced affinity for actin. In motility assays, 519–524/+A myosin released actin filaments upon addition of ATP and was unable to support movements. L596S myosin was also inactive, but in this case actin filaments stayed immobile even after the addition of ATP. Transient kinetic measurements demonstrated that 519–524/+A S1 is not only slower than wild type to bind actin filaments, but also slower to dissociate from actin filaments. Based on these results, we concluded that the 519–524 loop is not a major actin binding site but aids actin binding by facilitating a critical conformational change.     

Interactions of the RAD7 and RAD23 excision repair genes of Saccharomyces cerevisiae with DNA repair genes in different epistasis groups

Summary The RAD7 and RAD23 genes of S. cerevisiae affect the efficiency of excision repair of UV-damaged DNA. We have examined the UV survival of strains carrying the rad7 or rad23 deletion mutation in combination with deletion mutations in genes affecting different DNA repair pathways. As expected, the rad7 and rad23 mutations interact epistatically with the excision repair defective rad1 mutation, and synergistically with the rad6 and rad52 mutations that affect the postreplication repair and recombinational repair pathways, respectively. However, the rad7rad6 and the rad23rad6 mutants exhibit the same level of UV sensitivity as the radlrad6 mutant. This observation is of interest since, in contrast to the rad7 or the rad23 mutations, the rad1 mutant is very UV sensitive and highly excision defective. This observation suggests that RAD6 and RAD7 and RAD23 genes compete for the same substrate during DNA repair.     

Einfluß von Aldosteron auf den Natriumtransport in den Sammelrohren der Säugetierniere

Zusammenfassung Durch Mikropunktion und -perfusion der Vasa recta an der freigelegten Nierenpapille von Wistarratten wurde es möglich, die schwankenden Harnstoff- und Natriumkonzentrationen im Interstitium des Nierenmarks zu beseitigen und definierte Versuchsbedingungen für die Sammelrohre zu schaffen.An diesen funktionell isolierten Sammelrohrabschnitten wurdenin situ sowohl die Gleichgewichtskonzentrationsdifferenz bei fehlendem Nettosubstanz-und -volumenfluß (C _Na) als auch der Nettonatriumtransport ( _Na) bei gleicher Natriumkonzentration auf beiden Seiten der Sammelrohrwand gemessen. Es konnte gezeigt werden, daß unter diesen Versuchsbedingungen, bei denen die Tiere in Antidiurese sind, die Natriumrückresorption aus den Sammelrohren isoton abläuft.Die Versuche wurden an vier Tiergruppen durchgeführt: an adrenalektomierten Tieren, an normal ernährten Tieren, an salzarm ernährten Tieren und an normal ernährten Tieren, die zusätzlich Aldosteron bekamen. _{Na iso} war bei adrenalektomierten Tieren 1,2·10^–5 bei normal ernährten Tieren 3,1·10^–5, bei salzarm ernährten Tieren 4,1·10^–5 und bei normal ernährten Tieren unter Aldosteronsubstitution 4,2·10^–5 Äq·mm^–2·sec^–1. Die entsprechenden C _Na werte waren 4, 31, 98, 93 mÄq/l.Unter der Annahme, daß _{Na iso} die Transportkapazität des Systems angibt und daß C _Na bei gegebenem _{Na iso} umgekehrt proportional der Leckpermeabilität für Natriumionen ist, kann man aus den vorliegenden Daten schließen, daß Aldosteron am Sammelrohr nicht nur die innere Transportkapazität für Na erhöht, sondern auch die Leckpermeabilität für Na herabsetzt.     

Effects of the order of running and cycling of similar intensity and duration on pulmonary diffusing capacity in triathletes

To study the pathophysiological mechanisms involved in the decrease of post-triathlon diffusing capacity (DLco), blood rheologic properties (blood viscosity: _b; changes in plasma volume: PV) and atrial natriuretic factor (ANF) were assessed in ten triathletes during cycle-run (CR) and run-cycle (RC) trials at a metabolic intensity of 75% of maximal oxygen consumption (O_2max). The DLco was measured before and 10 min after trials. ANF and PV were measured at rest, after the cycle and run of CR and RC trials, and at the end of and 10 min after exercise. RC led to a greater DLco decrease, a lower ANF concentration and a lower PV than did CR, whereas for both CR and RC _b was increased throughout exercise and 10 min after. In addition, after CR the DLco decrease was inversely correlated (r=–0.764; P<0.01) with PV. The association of decreased plasma volume, increased _b, and lower ANF concentrations after RC suggested that lower blood pulmonary volume may have caused the greater decrease in Dlco as compared with CR. The inverse correlation between PV and DLco reinforces the hypothesis that fluid shifts limit the post-exercise DLco decrease after the CR succession in triathletes. Lastly, cycling in the crouched position might increase intra-thoracic pressure, decrease thorax volume due to the forearm position on the handlebars, and weaken peripheral muscular pump efficacy, all of which would limit venous return to the heart, and thus result in low pulmonary blood volume. Compared with cycling, running appeared to induce the opposite effects.