In vitro development of histotropic larvae of Oesophagostomum dentatum under various conditions of cultivation

We performed a total of 14 trials to evaluate the culture conditions suited best for the in vitro production of fourth-stage larvae (L4) of Oesophagostomum dentatum. Chicken embryo extract was shown to be redundant, whereas pig blood serum, trypticase, liver extract, and yeast extract proved to be important medium ingredients. Development to L4 could be moderately accelerated by increases of temperature (40 °C) and of CO₂ concentration (20% in air), but the total yield of L4 could not be improved. Thus, conditions of 38.5 °C and 10% CO₂ are recommended for routine application. Received: 15 June 1998 / Accepted: 9 July 1998     

Comparison of simultaneous pH measurements made with 8-hydroxypyrene-1,3,6-trisulphonic acid (HPTS) and pH-sensitive microelectrodes in snail neurones

 We have evaluated the pyrene-based ratiometric fluorescent dye, 8-hydroxypyrene-1,3,6-trisulphonic acid (HPTS), by using it in conjunction with glass pH-sensitive microelectrodes to measure intracellular pH (pH_i) in voltage-clamped snail neurones. Intracellular acidification with propionic acid, and alkalinization following the activation of H⁺ channels allowed the calibration of the dye to be compared with that of the pH microelectrode over the pH range 6.50–7.50. HPTS calibrated in vitro and glass pH-sensitive microelectrodes produced similar absolute resting pH_i values, 7.16±0.05 (n=10) and 7.17±0.06 (n=9) respectively in nominally CO₂/HCO₃ ^–-free saline. At both extremes of the pH range there were small discrepancies. At acidic pH_i, 6.87±0.09 (n=5), the intracellular HPTS measurement differed by –0.08±0.03 pH units from the pH-sensitive microelectrode measurement. At alkaline pH_i,7.32±0.10 (n=5), HPTS measurements produced pH values that differed by +0.07±0.04 pH units from those of the pH-sensitive microelectrode. Some of the discrepancy could be accounted for by the slow response of the recessed-tip pH-sensitive microelectrode (time constant 77±15 s, n=3). Further experiments showed that HPTS, used at an intracellular concentration of 200 μM to 2 mM, did not block activity-dependent pH_i changes. The intracellular HPTS concentration was calculated by measurement of intracellular chloride during a series of HPTS-KCl injections. Comparison of HPTS with 2′,7′-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF), at the same concentration, showed that HPTS produces a larger change in ratio over the pH range 6.00–8.00. Received: 19 February 1998 / Received after revision and accepted: 22 April 1998     

Partial anaerobiosis induces infectivity of Leishmania infantum promastigotes

Leishmania infantum stationary-phase promastigotes could acquire infectivity via preincubation in a partially anaerobic medium (95% air/5% CO₂) for 16 h before the infection, whereas promastigotes were efficiently destroyed when no CO₂ was present. Incubation of L. infantum promastigotes with additional glucose (20 and 50 mM) greatly increased infection parameters in the absence of CO₂; this is consistent with a “reverse Pasteur effect.” Results showed that culture at 33 °C permitted survival and amastigote multiplication (a nearly 10-fold increase in amastigotes as compared with those observed in 37 °C cultures). This finding was obtained with the two strains of L. infantum tested (Doba and PB75). Received: 28 November 1998 / Accepted: 17 December 1998     

Enhanced cerebral CO2 reactivity during strenuous exercise in man

Light and moderate exercise elevates the regional cerebral blood flow by ~20% as determined by ultrasound Doppler sonography (middle cerebral artery mean flow velocity; MCA V _mean). However, strenuous exercise, especially in the heat, appears to reduce MCA V _mean more than can be accounted for by the reduction in the arterial CO₂ tension (P _aCO₂). This study evaluated whether the apparently large reduction in MCA V _mean at the end of exhaustive exercise relates to an enhanced cerebrovascular CO₂ reactivity. The CO₂ reactivity was evaluated in six young healthy male subjects by the administration of CO₂ as well as by voluntary hypo- and hyperventilation at rest and during exercise with and without hyperthermia. At rest, P _aCO₂ was 5.1±0.2 kPa (mean ± SEM) and MCA V _mean 50.7±3.8 cm s⁻¹ and the relationship between MCA V _mean and P _aCO₂ was linear (double-log slope 1.1±0.1). However, the relationship became curvilinear during exercise (slope 1.8±0.1; P<0.01 vs. rest) and during exercise with hyperthermia (slope 2.3±0.3; P<0.05 vs. control exercise). Accordingly, the cerebral CO₂ reactivity increased from 30.5±2.7% kPa⁻¹ at rest to 61.4±10.1% kPa⁻¹ during exercise with hyperthermia (P<0.05). At exhaustion P _aCO₂ decreased 1.1±0.2 kPa during exercise with hyperthermia, which, with the determined cerebral CO₂ reactivity, accounted for the 28±10% decrease in MCA V _mean. The results suggest that during exercise changes in cerebral blood flow are dominated by the arterial carbon dioxide tension.     

Metabolism in rats during antiorthostatic hypokinesia

O₂ consumption and CO₂ release in 3 groups of awake rats were studied on a MM-100 metabolic monitor system (CWE Inc.). The animals of 2 groups were preadapted to 4-h maintenance in special boxes (2 weeks). The rats could perform rotational movements and limited movements in the rostrocaudal direction (hypokinesia). The animals of one group were daily exposed to 4-h antiorthostatic load (≤45°) for 2 weeks. After 2 weeks, the intensity of metabolism in rats with antiorthostatic hypokinesia was lower than in hypokinetic specimens (by 15–20%, p<0.05) and freely moving animals (by 20–25%, p<0.05). Interleukin-6 concentration in rats with antiorthostatic hypokinesia (0.25±0.09 pg/ml) was lower than in hypokinetic (4.01±0.57 pg/ml) and freely moving animals (3.69±0.56 pg/ml). The decrease in the concentration of a proinflammatory cytokine interleukin-6 during experimental antiorthostatic hypokinesia reflects inhibition of metabolic processes, which are activated during antiorthostatism (but not hypokinesia). __________ Translated from Byulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 146, No. 7, pp. 42–45, July, 2008     

Antileishmanial potential of a marine sponge, <Emphasis Type="Italic">Haliclona exigua</Emphasis> (Kirkpatrick) against experimental visceral leishmaniasis

In this study, we are reporting antileishmanial activity of a marine sponge Haliclona exigua, belonging to phylum Porifera. The crude methanol extract and its three fractions were tested both in vitro and in vivo. The crude extract exerted almost complete inhibition of promastigotes at 50 μg/ml and 76.4 ± 6.5% inhibition of intracellular amastigotes at 100 μg/ml concentration with IC₅₀ values of 18.6 μg/ml and 47.2 μg/ml, respectively. When administered to Leishmania donovani infected hamsters at a dose of 500 mg/kg × 5, p.o., it resulted in 72.2 ± 10.4% inhibition of intracellular amastigotes. At a lower dose (250 mg/kg), it exhibited 43.9 ± 5.1% inhibition. Among the fractions, highest antileishmanial activity both in vitro (>90%) and in vivo (60.9 ± 18.3%) was observed in n-butanol (soluble) fraction with IC₅₀ values of 8.2 μg/ml and 31.2 μg/ml against promastigotes and intracellular amastigotes, respectively. Hexane fraction also showed comparatively good activity against both the stages of parasites in vitro but was moderately active in leishmania-infected hamsters. Chloroform fraction resulted in 45 ± 10.2% inhibition in vivo at a dose of 500 mg/kg × 5, p.o., whereas it was inactive in vitro. n-Butanol (insoluble) fraction was inactive both in vitro and in vivo. Araguspongin C, an alkaloid isolated from n-butanol (soluble) fraction exhibited moderate inhibition of promastigotes and intracellular amastigotes at 100 μg/ml but showed weak antileishmanial action in vivo. Our findings indicate that this marine sponge has the potential to provide new lead toward development of an effective antileishmanial agent and, hence, calls for more exhaustive studies for exploiting the vast world of marine resources to combat the scourge of several parasitic diseases.     

Accuracy and reliability of the ParvoMedics TrueOne 2400 and MedGraphics VO2000 metabolic systems

This study examined the accuracy and reliability of the MedGraphics VO2000 (VO2000) portable metabolic system and the ParvoMedics TrueOne 2400 (TrueOne 2400) metabolic cart against the criterion Douglas bag (DB) method. Ten healthy males (age 20 ± 1.7 years) had their gas exchange variables measured at rest and during cycling at 50, 100, 150, 200, and 250 W. Each stage was 10–12 min. For half of the stage gas exchange was measured with the DB and TrueOne 2400 simultaneously and for the other half of the stage gas exchange was measured with the VO2000. The testing was performed on two separate days and the order in which the equipment was used in each stage was randomized. Reliability between days for V _E (CV 7.3–8.8%) was similar among devices, however, for VO₂, and VCO₂ the VO2000 (CV 14.2–15.8%) was less reliable compared to the DB (CV 5.3–6.0%) and TrueOne 2400 (CV 4.7–5.7%). The TrueOne 2400 was not significantly different from the DB at rest or any work rate for V _E, VO₂, or VCO₂ (P ≥ 0.05). The VO2000 was significantly different from the DB for V _E at 50–100 W, VO₂ at rest and 100–250 W, and VCO₂ at rest and 200–250 W (all, P < 0.05). The TrueOne 2400 provides accurate and reliable results for the measurement of gas exchange variables. The VO2000 portable metabolic system was less reliable for measuring VO₂ and VCO₂ and generally overestimates VO₂ at most cycling work rates. Further research is needed to confirm the results found with the VO2000.     

Influence of sildenafil on lung diffusion during exposure to acute hypoxia at rest and during exercise in healthy humans

We sought to determine the influence of sildenafil on the diffusing capacity of the lungs for carbon monoxide (DL_CO) and the components of DL_CO (pulmonary capillary blood volume V _c, and alveolar–capillary membrane conductance D _M) at rest and following exercise with normoxia and hypoxia. This double-blind placebo-controlled, cross-over study included 14 healthy subjects (age = 33 ± 11 years, ht = 181 ± 8 cm, weight = 85 ± 14 kg, BMI = 26 ± 3 kg/m², peak normoxic VO₂ = 36 ± 6 ml/kg, mean ± SD). Subjects were randomized to placebo or 100 mg sildenafil 1 h prior to entering a hypoxic tent with an FiO₂ of 12.5% for 90 min. DLCO, V _c, and D _M were assessed at rest, every 3 min during exercise, at peak exercise, and 10 and 30 min post exercise. Sildenafil attenuated the elevation in PAP at rest and during recovery with exposure to hypoxia, but pulmonary arterial pressure immediately post exercise was not different between sildenafil and placebo. Systemic O₂ saturation and VO_2peak did not differ between the two conditions. DL_CO was not different between groups at any time point. V _C was higher with exercise in the placebo group, and the difference in D _M between sildenafil and placebo was significant only when corrected for changes in V _c (D _M/V _c = 0.57 ± 0.29 vs. 0.41 ± 0.16, P = 0.04). These results suggest no effect of sildenafil on DLCO, but an improvement in D _M when corrected for changes in V _c during short-term hypoxic exposure with exercise.     

Carbon monoxide inhibits hypoxic pulmonary vasoconstriction in rats by a cGMP-independent mechanism

 Hypoxia activates erythropoietin-producing cells, chemoreceptor cells of the carotid body and pulmonary artery smooth muscle cells (PSMC) with a comparable arterial PO₂ threshold of some 70 mmHg. The inhibition by CO of the hypoxic responses in the two former cell types has led to the proposal that a haemoprotein is involved in the detection of the PO₂ levels. Here, we report the effect of CO on the hypoxic pulmonary vasoconstriction (HPV). Pulmonary arterial pressure (PAP) was measured in an in situ, blood-perfused lung preparation. PAP in normoxia (20% O₂, 5% CO₂) was 15.2±1.8 mmHg, and hypoxia (2% O₂, 5% CO₂) produced a ΔPAP of 6.3±0.4 mmHg. Addition of 8% or 15% CO to the hypoxic gas mixture reduced the ΔPAP by 88.3±2.7% and 78.2±6.1% respectively. The same levels of CO did not affect normoxic PAP nor reduced the ΔPAP produced by angiotensin II. The effect of CO was studied after inhibition of the NO-cyclic guanosine monophosphate (cGMP) cascade with N-methyl-l-arginine (5·10^–5 M) or methylene blue (1.4·10^–4 M). It was found that both inhibitors more than doubled the hypoxic ΔPAP without altering the effectiveness of CO to inhibit the HPV. In in vitro experiments we verified the inhibition of guanylate cyclase by measuring the levels of cGMP in segments of the pulmonary artery. Cyclic GMP levels were 1.4±0.2 (normoxia), 2.5±0.3 (hypoxia) and 3.3±0.5 pmole/mg tissue (hypoxia plus 8% CO); sodium nitroprusside increased normoxic cGMP levels about fourfold. Methylene blue reduced cGMP levels to less than 10% in all cases, and abolished the differences among normoxic, hypoxic and hypoxic plus CO groups. It is concluded that CO inhibits HPV by a NO-cGMP independent mechanism and it is proposed that a haemoprotein could be involved in O₂-sensing in PSMC. Received: 17 March 1997 / Received after revision: 10 June 1997 / Accepted: 11 July 1997     

A transient dilatation of pressurised rat cerebral arteries during rapid pressure increases is mediated by nitric oxide

 In pressurised resistance arteries in vitro, rapid pressure increases cause a transient ”peak” dilatation, followed by a myogenic constriction. The mechanism of transient dilatation was investigated in isolated rat cerebral arteries in vitro using pressure myography. The peak increased with the amplitude of the pressure step. A near-maximal dilatation to 118±1.6% (SEM, n=20) of the diameter at 30 mmHg was produced by pressure steps from 30 to 75 mmHg. N ^ω-nitro-l-arginine methyl ester (L-NAME, 20 μM) depressed the peak at the onset of a 30 to 75 mmHg pressure step to 49.8±14% of the control (n=6; P=0.04). D-NAME (20 μM) had no significant effect (82.1±13%; n=4; P=0.13). l-Arginine (400 μM) enhanced the peak (164±17% of control; n=8; P=0.01). Oxadiazolol (4,3-a) quinoxalin-1-one (ODQ, 2 μM) depressed the peak to 33.2±12% of control (n=5; P=0.012). 6-Anilino-5,8-quinolinedone (LY 83583, 10 μM) depressed the peak to 18.8±2.9% of control (n=3; P=0.01). Removing the endothelium decreased the peak to 15.3±11% of control (n=3; P=0.04). In conclusion, in rat cerebral arteries, the initial dilatation at the onset of a rapid step increase in pressure is an active dilatation involving endothelial NO release. Received: 10 November 1997 / Received after revision: 19 January 1998 / Accepted: 26 February 1998     

The role of muscle pump in the development of cardiovascular drift

This study examined the role of muscle pump in the development of cardiovascular drift (CV_drift) during cycling. Twelve healthy males (23.4 ± 0.5 years, mean ± SE) exercised for 90 min with 40 and 80 pedal revolutions per minute (rpm) at the same oxygen consumption, in two separate days. CV_drift was developed in both conditions as indicated by the drop in stroke volume (SV) and the rise in heart rate (HR) from the 20th min onwards (ΔSV = −16.2 ± 2.0 and −17.1 ± 1.0 ml beat⁻¹; ΔHR = 18.3 ± 2.0 and 17.5 ± 3.0 beats min⁻¹ for 40 and 80 rpm, respectively, P < 0.05) but without difference between conditions. Mean cardiac output (CO₂ rebreathing) was 14.7 ± 0.3 l min⁻¹ and 15.0 ± 0.3 l min⁻¹, and mean arterial pressure was 100.0 ± 1.0 mmHg and 96.7 ± 0.8 mmHg for 40 and 80 rpm, respectively, without significant changes over time, and without difference between conditions. Electromyographic activity (iEMG) was lower throughout exercise with 80 rpm (35.6 ± 1.2% and 11.0 ± 1.0% for 40 and 80 rpm, respectively). Similarly, total hemoglobin, determined with near-infrared spectroscopy (NIRS) was 58.0 ± 0.8 (AU) for 40 rpm and 53.0 ± 1.4 (arbitrary units) for 80 rpm, from 30th min onwards (P < 0.05), an indication of lower leg blood volume during the faster pedal rate condition. Thermal status (rectal and mean skin temperature), blood and plasma volume changes, blood lactate concentration, muscle oxygenation (NIRS signal) and the rate of perceived exertion were similar in the two trials. It seems that muscle pump is not an important factor for the development of CV_drift during cycling, at least under the present experimental conditions.     

Expression of low-density lipoprotein receptors in peripheral blood and tonsil B lymphocytes

B lymphocytes, purified from peripheral leucocytes from young normolipaemic humans, expressed and internalized low-density lipoprotein receptors (LDLR). The expression was assessed by a monoclonal anti-LDLR. The internalization of LDL was assessed by LDL labelled with ¹²⁵I (¹²⁵I-LDL) and 1,1′-dioctadecyl-3,3,3′,3′ tetramethyl-indocarboxycyanine perchlorate (LDL-DiI). The expression of LDLR, assessed by anti-LDLR, was: 38 ± 8% (n = 5) for fresh purified cells, 60 ± 10% (n = 12) for non-stimulated cells, 79 ± 5% (n = 10) for IL-2 (100 U/ml)-stimulated cells and 95 ± 5% (n = 8) for pokeweed mitogen (PWM) (1:200 dilution)-stimulated cells. The optimal concentrations of agonist were 100 U/ml of IL-2, and 1:200 dilution of PWM. IL-2 and PWM increased the internalization of LDL-DiI by 1.5-fold. The internalization of LDL-DiI was maximal at 60 μg of protein/ml (48 ± 8%). Scatchard analysis revealed a Kd of 3.2 ± 0.22 × 10^?8 M and 2180 ± 190 binding sites in non-stimulated cells, a Kd of 7.73 ± 0.36 × 10^?9 M and 12 500 ± 430 binding sites for IL-2 (100 U/ml)-stimulated cells, and a Kd of 7.2 ± 0.43 × 10^?9 M and 13 250 ± 450 binding sites for PWM (1:200 dilution)-stimulated cells. Lineweaver–Burk analysis of LDL binding (LDL-DiI) revealed that the apparent Kd for non-stimulated cells was 1.3 ± 0.11 × 10^?8 M , and 9.2 ± 0.2 × 10^?9 M and 7.5 ± 0.25 × 10^?9 M for IL-2- and PWM-stimulated cells, respectively. B lymphocytes from tonsils also showed a high expression of LDLR assessed with anti-LDLR (70 ± 6%). The high expression of LDLR and the avid internalization of LDL suggest that LDL may be important for B cell physiological responses.     

Reliability of a cycling time trial in a glycogen-depleted state

The aim of this study was to investigate the reliability of a protocol designed to simulate endurance performance in events of long duration (∼5 h) where endogenous carbohydrate stores are low. Seven male subjects were recruited (age 27 ± 7 years, VO_2max 66 ± 5 ml/kg/min, W _max 367 ± 42 W). The subjects underwent three trials to determine the reliability of the protocol. For each trial subjects entered the laboratory in the evening to undergo a glycogen-depleting exercise trial lasting approximately 2.5 h. The subjects returned the following morning in a fasted state to undertake a 1-h steady-state ride at 50% W _max followed by a time trial of approximately 40-min duration. Each trial was separated by 7–14 days. The trials were analysed for reliability of time to completion of the time trial using a coefficient of variation (CV), with 95% confidence intervals (data are mean ± SD). The times to complete the three trials were 2,546 ± 529, 2,585 ± 490 and 2,568 ± 555 s for trials 1, 2 and 3, respectively. The CV between trials 1 and 2 was 4.5% (95% CI 2.9–10.4%) and between trials 2 and 3, 3.8% (95% CI 2.4–9.9%). There was no difference in oxygen uptake, respiratory exchange ratio, carbohydrate oxidation, fat oxidation, plasma glucose concentration and plasma lactate concentration between the three trials. Therefore we can conclude that prior glycogen depletion does produce a reliable measure of performance with metabolic characteristics similar to ultraendurance exercise.     

Evaluation of antileishmanial potential of Tinospora sinensis against experimental visceral leishmaniasis

The chemotherapeutic interventions against visceral leishmaniasis (VL) are limited and facing serious concerns of toxicity, high cost, and emerging drug resistance. There is a greater interest in new drug developments from traditionally used medicinal plants which offers unprecedented diversity in structures and bioactivity. With this rationale, ethanolic extract of Tinospora sinensis Linn and its four fractions were tested in vitro against promastigotes and intracellular amastigotes and in vivo in Leishmania donovani infected hamsters. Ethanolic extract exhibited an appreciable activity against promastigotes (IC₅₀ 37.6 ± 6.2 μg/ml) and intracellular amastigotes (IC₅₀ 29.8 ± 3.4 μg/ml). In hamsters, it resulted in 76.2 ± 9.2% inhibition at 500 mg/kg/day × 5 oral dose level. Among fractions, n-butanol imparted highest in vitro and in vivo activities. Ethanolic extract and butanol fraction also enhances reactive oxygen species (ROS) and nitric oxide (NO) release. The results indicate that T. sinensis may provide new lead molecules for the development of alternative drugs against VL.     

Validity of the Oxycon Mobile metabolic system under field measuring conditions

Abstract  

This study aimed to validate a portable metabolic system in field measuring conditions, such as prolonged moderate exercise at low temperatures, high humidity and with external wind. VO₂, VCO₂, RER and V _E were measured using the Oxycon Mobile (OM), with a windshield, during cycle ergometer exercise: (1) indoors at three submaximal workloads with no wind or with external wind (13–20 m s⁻¹) from front, side and back; (2) at two submaximal workloads outdoors (12 ± 2°C; 86 ± 7% relative humidity (RH)), with and without a system for drying the ambient air around the air sampling tube; and (3) at one workload outdoors for 45 min (5 ± 4°C; 69 ± 16.5% RH). Any physiological drift was checked for with pre- and postmeasurements by the Douglas bag method (DBM). A minor effect of external wind from behind was noted in RER and V _E (−2 and −3%). The system for drying the ambient air around the gas sampling tube had no effect on the measured levels. A small difference in VCO₂ drift between the OM and DBM (1.5 mL min⁻²) was noted in the stability test. The results indicated that heavy external wind applied from different directions generally does not affect the measurements of the OM and further that, when using a unit for drying the ambient air around the gas sampling tube, the OM can accurately measure VO₂, RER and V _E at submaximal workloads for at least 45 min under challenging conditions with regard to humidity and temperature.     

Role of PKC in the effects of α1-adrenergic stimulation on Ca2+ transients, contraction and Ca2+ current in guinea-pig ventricular myocytes

 The effects of α₁-adrenoceptor stimulation on intracellular Ca²⁺ transients, contractility and L-type Ca²⁺ current (I _Ca,L) were studied in single cells isolated from ventricles of guinea-pig hearts. The aim of our study was to elucidate the mechanisms of the positive inotropic effect of α₁-adrenergic stimulation by focussing on the role of protein kinase C (PKC). Phenylephrine, an α₁-adrenergic agonist, at concentrations of 50–100 μM elicited a biphasic inotropic response: a transient negative inotropic response (22.9±6.0% of control) followed by a sustained positive inotropic response (61.0±8.4%, mean±SE, n=12). The Ca²⁺ transient decreased by 10.2±3.9% during the negative inotropic phase, while it increased by 67.7±10% (n=12) during the positive inotropic phase. These effects were inhibited by prazosin (1 μM), a α₁-adrenergic antagonist. Phenylephrine increased the I _Ca,L by 60.8±21% (n=5) during the positive inotropic phase. To determine whether activation of PKC is responsible for the increases in Ca²⁺ transients, contractile amplitude and I _Ca,L during α₁-adrenoceptor stimulation, we tested the effects of 4β-phorbol 12-myristate 13-acetate (PMA), a PKC activator, and of bisindolylmaleimide I (GF109203X) and staurosporine, both of which are PKC inhibitors. PMA mimicked phenylephrine’s effects on Ca²⁺ transients, contractile amplitude and I _Ca,L. PMA (100 nM) increased the Ca²⁺ transient, contractile amplitude and I _Ca,L by 131±17%, 137±25% (n=8), and 81.1±26% (n=5), respectively. Prior exposure to GF109203X (1 μM) or staurosporine (10 nM) prevented the phenylephrine-induced increases in Ca²⁺ transients, contractile amplitude and I _Ca,L. Our study suggests that during α₁-adrenoceptor stimulation increase in I _Ca,L via PKC causes an increase in Ca²⁺ transients and thereby in the contractile force of the ventricular myocytes. Received: 16 July 1998 / Received after revision and accepted: 20 October 1998     

The effect of phosphate on the relaxation of frog skeletal muscle

 The effect of phosphate on the relaxation of isometrically contracting single skinned fibres from the semitendinosus muscle of the frog Rana temporaria has been investigated using laser pulse photolysis of the photolabile caged calcium-chelator diazo-2 to rapidly reduce the Ca²⁺ (<2 ms) within the fibre and produce >90% relaxation of force. Relaxation occurred in two phases – an initial linear shoulder which lasted approximately 20 ms followed by a double-exponential phase which gave two rate constants, k ₁ (43.4±1.8 s^–1, mean ±SEM, n=14) and k ₂ (15.6±0.3 s^–1, mean ±SEM, n=14) at 12°C. Increased phosphate concentrations did not affect the linear phase, but slowed the double-exponential phase following photolysis of diazo-2 in a dose-dependent fashion (k ₅₀= 0.9 mM for k ₁, 1.15 mM for k ₂). Reducing the concentration of contaminating phosphate (from 640 μM to 100 μM) led to an increase in the rate of the double-exponential phase (k ₁=67.1±4.4 s^–1, k ₂=19.7±0.6 s^–1, mean ±SEM, n=12). Time-resolved measurements of sarcomere length during relaxation, both in control fibres and in the presence of a raised phosphate concentration, reveal a <2% change throughout the whole relaxation transient, and less than 0.1% at the end of the linear phase. This finding implies that gross changes in sarcomere length do not contribute to the decay of the relaxation transient seen upon diazo-2 photolysis. Our results suggest that cross-bridges in states prior to phosphate release are already committed to force generation and must relax by releasing phosphate, rather than by a reversal of the force-generating step to a weakly bound, low-force phosphate-bound state. These findings also indicate that an increase in the phosphate concentration within muscle fibres plays an important part in the slowing of relaxation observed in skeletal muscle fatigue and that the relaxation transients observed upon diazo-2 photolysis represent a disengagement of the cross-bridges. Received: 14 September 1998 / Received after revision and accepted: 20 October 1998     

Influence of postnatal changes in action potential duration on Na-Ca exchange in rabbit ventricular myocytes

 Cardiac Na-Ca exchanger (NCX) expression and current density are significantly greater in newborn rabbit hearts compared with adults. However, the relatively short action potential (AP) at birth may limit the impact of increased NCX expression by diminishing Ca²⁺ entry via Na-Ca exchange current (I _NaCa). To address the interdependence of AP duration and NCX activity, we voltage-clamped newborn (NB, 1–5 day), juvenile (JV, 10–14 day) and adult (AD) rabbit myocytes with a series of APs of progressively increasing duration (APD₉₀: 108–378 ms) under nominally chloride-free conditions. In each age group we quantified an increase in outward (Q _Exout) and inward (Q _Exin) Ni²⁺-sensitive charge movement in response to AP prolongation. Q _Exout and Q _Exin measured during age-appropriate APs declined postnatally [Q _EXout: NB (2 day) 0.19 ± 0.02, JV (10 day) 0.10 ± 0.01, AD 0.04 ± 0.002; Q _EXin: NB –0.2 ± 0.01, JV –0.11 ± 0.02; AD –0.04 ± 0.003 pC/pF] despite the significantly shorter APD₉₀ of newborn myocytes (NB 122 ± 10; AD 268 ± 22 ms). When Ca²⁺ fluxes by other transport pathways were blocked with nifedipine, ryanodine and thapsigargin, age-appropriate APs elicited contractions in NB and JV but not AD myocytes (NB 4.8 ± 0.5, JV 1.2 ± 0.3% resting length). These data demonstrate that a shorter AP does not negate the impact of increased NCX expression at birth. Received: 23 September 1997 / Received after revision: 2 January 1998 / Accepted: 5 January 1998     

Non-invasive assessment of cardiac output and stroke volume in patients during exercise

Summary A one-step CO₂ rebreathing method for the determination of cardiac output and stroke volume (SV) has been evaluated by comparison with the direct Fick technique during recumbent exercise (10–90 W) in 13 patients. In an initial analysis, the influence of different rebreathing times and of correction for haemoglobin concentration was studied. The best correlation with the direct Fick technique was obtained with the longest analysis time, i. e. 21 s, and correction for variations in haemoglobin concentration further improved the correlation. Consequently, an analysis time of 21 s and correction for haemoglobin have been used. At low cardiac outputs, the CO₂-rebreathing method overestimated the flow compared to the Fick technique. The correlation between the methods, however, was so good that a valid estimate of cardiac output could be obtained from the CO₂ rebreathing method with appropriate corrections (Cardiac output, CO₂ method=2.7+0.77. Cardiac output, Fick; r=0.91; Residual Standard deviation (SD res) =0.77 l · min⁻¹). Stroke volumes measured with the CO₂ rebreathing method did not differ significantly from those obtained with the direct Fick technique, although there was a tendency to overestimate stroke volume with the CO₂ rebreathing method (SV, CO₂ method=12+0.89 · SV, Fick; r=0.82; SD res=11 ml).