Estimation of cardiomyocyte transmembrane potential with potential-sensitive fluorescent probes

Transmembrane potentials on the plasma (Δφ₄) and mitochondrial (Δφ_m) membrane of isolated rat cardiomyocytes were estimated using the potential-sensitive fluorescent probe DSM. The values were −93±4 and −196±11 mV, respectively. Sufan significantly decreased the reduction of Δφ_m induced by chemical hypoxia. The effects of antiarrhythmic drugs on changes in Δφ_p were studied using the fluorescent probe dis-C₃-(5). Lidocaine, novocainamide, richlocaine, and leocaine blocked depolarization of the myocyte plasma membrane induced by electrical stimulation and did not affect the Δφ_m. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 126, No. 11, pp. 594–597, November, 1998     

cAMP-dependent inward rectifier current in neurons of the rat suprachiasmatic nucleus

Electrophysiological properties of the inward rectification of neurons in the rat suprachiasmatic nucleus (SCN) were examined by using the single-electrode voltage-clamp method, in vitro. Inward rectifier current (I _H) was produced by hyperpolarizing step command potentials to membrane potentials negative to approximately −60 mV in nominally zero-Ca²⁺ Krebs solution containing tetrodotoxin (1 μM), tetraethylammonium (40 mM), Cd²⁺ (500 μM) and 4-aminopyridine (1 mM).I _H developed during the hyperpolarizing step command potential with a duration of up to 5 s showing no inactivation with time.I _H was selectively blocked by extracellular Cs⁺ (1 mM). The activation of the H-channel conductance (G _H) ranged between −55 and −120 mV. TheG _H was 80–150 pS (n=4) at the half-activation voltage of −84±7 mV (n=4). The reversal potential ofI _H obtained by instantaneous current voltage (I/V) relations was −41±6mV (n=4); it shifted to −51±8mV (n=3) in low-Na⁺ (20 mM) solution and to −24±4 mV (n=4) in high-K⁺ (20 mM) solution. Forskolin (1–10 μM) produced an inward current and increased the amplitude ofI _H. Forskolin did not change the half-activation voltage ofG _H. 8-Bromo-adenosine 3′,5′-cyclic monophosphate (8-Br-cAMP, 0.1–1 mM) and dibutyryl-cAMP (0.1–1 mM) enhancedI _H. 3-Isobutyl-1-methylxanthine (IBMX, 1 mM) also enhancedI _H. The results suggest that the inward rectifier cation current is regulated by the basal activity of adenylate cyclase in neurons of the rat SCN.     

Mechanisms of reduced mitochondrial Ca2+ accumulation in failing hamster heart

Mitochondrial Ca²⁺ plays important roles in the regulation of energy metabolism and cellular Ca²⁺ homeostasis. In this study, we characterized mitochondrial Ca²⁺ accumulation in Syrian hamster hearts with hereditary cardiomyopathy (strain BIO 14.6). Exposure of isolated mitochondria from 70 nM to 30 μM Ca²⁺ ([Ca²⁺]_o) caused a concentration-dependent increase in intramitochondrial Ca²⁺ concentrations ([Ca²⁺]_m). The [Ca²⁺]_m was significantly lower in cardiomyopathic (CMP) hamsters than in healthy hamsters when [Ca²⁺]_o was higher than 1 μM and a decrease of about 52% was detected at [Ca²⁺]_o of 30 μM (916 ± 67 nM vs 1,932 ± 132 nM in control). A possible mechanism responsible for the decreased mitochondrial Ca²⁺ uptake in CMP hamsters is the depolarization of mitochondrial membrane potential (Δψ _m). Using a tetraphenylphosphonium (TPP⁺) electrode, the measured Δψ _m in failing heart mitochondria was −136 ± 1.5 mV compared with −159 ± 1.3 mV in controls. Analyses of mitochondrial respiratory chain demonstrated a significant impairment of complex I and complex IV activities in failing heart mitochondria. In summary, a less negative Δψ _m resulting from defects in the respiratory chain may lead to attenuated mitochondrial Ca²⁺ accumulation, which in turn may contribute to the depressed energy production and myocardial contractility in this model of heart failure. In addition to other known impairments of ion transport in sarcoplasmic reticulum and plasma membrane, results from this paper on mitochondrial dysfunctions expand our understanding of the molecular mechanisms leading to heart failure.     

The Plasmodium falciparum-induced anion channel of human erythrocytes is an ATP-release pathway

Infection with the malaria parasite Plasmodium falciparum induces osmolyte and anion channels in the host erythrocyte membrane involving ATP release and autocrine purinergic signaling. P. falciparum-parasitized but not unstimulated uninfected erythrocytes released ATP in a 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB; 7 μM)-sensitive and serum album (SA; 0.5% w/v)-stimulated manner. Since Plasmodium infection of human erythrocytes induces SA-dependent outwardly (OR) and SA-independent inwardly rectifying (IR) anion conductances, we tested whether the infection-induced OR channels directly generate an ATP release pathway. P. falciparum-parasitized erythrocytes were recorded in whole-cell mode with either Cl⁻ or ATP as the only anion in the bath or pipette. In parasitized cells with predominant OR activity, replacement of bath NaCl by Na–ATP (NMDG–Cl pipette solution) shifted the current reversal potential (V _rev) from −2 ± 1 to +51 ± 3 mV (n = 15). In cells with predominant IR activity, in contrast, the same maneuver induced a shift of V _rev to significantly larger (p ≤ 0.05, two-tailed t test) values (from −3 ± 1 to +66 ± 8 mV; n = 5) and an almost complete inhibition of outward current. The anion channel blocker NPPB reversibly decreased the ATP-generated OR currents from 1.1 ± 0.1 nS to 0.2 ± 0.05 nS and further shifted V _rev to +87 ± 7 mV (n = 12). The NPPB-sensitive fraction of the OR reversed at +48 ± 4 mV suggesting a relative permeability of P _ATP/P _Cl ≈ 0.01. Together, these data raise the possibility that the OR might be the electrophysiological correlate of an erythrocyte ATP release pathway. Canan Akkaya and Ekaterina Shumilina contributed equally to this work and, thus, share first authorship.     

Thermodynamic properties of hyperpolarization-activated current (Ih) in a subgroup of primary sensory neurons

Ih is a poorly selective cation current that activates upon hyperpolarization, present in various types of neurons. Our aim was to perform a detailed thermodynamic analysis of Ih gating kinetics, in order to assess putative structural changes associated with its activation and deactivation. To select dorsal root ganglia neurons that exhibit large Ih, we applied a current signature method by Petruska et al. (J Neurophysiol 84:2365–2379, 2000) and found appropriate neurons in cluster 4. Currents elicited by 3,000-ms hyperpolarizing pulses at 25 and 33°C were fitted with double exponential functions, yielding time constants similar to those of HCN1. The fast activation and deactivation rates showed temperature coefficients (Q ₁₀) of 2.9 and 3.1, respectively, while Q ₁₀ of the absolute conductance was 1.3. Using the Arrhenius–Eyring formalism we computed heights of voltage-independent Gibbs free energy and entropy barriers for each rate. The free energy barriers of the fast rates were just ∼2RT units lower than those of the corresponding slow rates (31.3 vs. 33.2RT for activation, and 24.7 vs. 25.8RT for deactivation, at 25°C). Interestingly, the entropy barriers of the slow rates were negative: −15.2R units for activation and −11.9R units for deactivation, compared to 4.6 and 1.3R units, respectively, for the fast component. The equivalent gating charge (z _g) (3.75 ± 0.32, mean ± SEM, at 25°C) and half-activation potential (V _1/2) (−70.0 ± 1.3 mV at 25°C) did not vary significantly with temperature.     

Is the balance between skeletal muscular metabolic capacity and oxygen supply capacity the same in endurance trained and untrained subjects?

We attempted to test whether the balance between muscular metabolic capacity and oxygen supply capacity in endurance-trained athletes (ET) differs from that in a control group of normal physically active subjects by using exercises with different muscle masses. We compared maximal exercise in nine ET subjects [Maximal oxygen uptake (VO₂max) 64 ml kg⁻¹ min⁻¹ ± SD 4] and eight controls (VO₂max 46 ± 4 ml kg⁻¹ min⁻¹) during one-legged knee extensions (1-KE), two-legged knee extensions (2-KE) and bicycling. Maximal values for power output (P), VO₂max, concentration of blood lactate ([La⁻]), ventilation (VE), heart rate (HR), and arterial oxygen saturation of haemoglobin (SpO₂) were registered. P was 43 (2), 89 (3) and 298 (7) W (mean ± SE); and VO₂max: 1,387 (80), 2,234 (113) and 4,115 (150) ml min⁻¹) for controls in 1-KE, 2-KE and bicycling, respectively. The ET subjects achieved 126, 121 and 126% of the P of controls (p < 0.05) and 127, 124, and 117% of their VO₂max (p < 0.05). HR and [La⁻] were similar for both groups during all modes of exercise, while VE in ET was 147 and 114% of controls during 1-KE and bicycling, respectively. For mass-specific VO₂max (VO₂max divided by the calculated active muscle mass) during the different exercises, ET achieved 148, 141, and 150% of the controls’ values, respectively (p < 0.05). During bicycling, both groups achieved 37% of their mass-specific VO₂ during 1-KE. Finally we conclude that ET subjects have the same utilization of the muscular metabolic capacity during whole body exercise as active control subjects.     

Regulation of membrane excitability by intracellular pH (pHi) changers through Ca2+-activated K+ current (BK channel) in single smooth muscle cells from rabbit basilar artery

Employing microfluorometric system and patch clamp technique in rabbit basilar arterial myocytes, regulation mechanisms of vascular excitability were investigated by applying intracellular pH (pH_i) changers such as sodium acetate (SA) and NH₄Cl. Applications of caffeine produced transient phasic contractions in a reversible manner. These caffeine-induced contractions were significantly enhanced by SA and suppressed by NH₄Cl. Intracellular Ca²⁺ concentration ([Ca²⁺]_i) was monitored in a single isolated myocyte and based the ratio of fluorescence using Fura-2 AM (R _340/380). SA (20 mM) increased and NH₄Cl (20 mM) decreased R _340/380 by 0.2 ± 0.03 and 0.1 ± 0.02, respectively, in a reversible manner. Caffeine (10 mM) transiently increased R _340/380 by 0.9 ± 0.07, and the ratio increment was significantly enhanced by SA and suppressed by NH₄Cl, implying that SA and NH₄Cl may affect [Ca²⁺]_i (p < 0.05). Accordingly, we studied the effects of SA and NH₄Cl on Ca²⁺-activated K⁺ current (IK_Ca) under patch clamp technique. Caffeine produced transient outward current at holding potential (V _h) of 0 mV, caffeine induced transient outward K⁺ current, and the spontaneous transient outward currents were significantly enhanced by SA and suppressed by NH₄Cl. In addition, IK_Ca was significantly increased by acidotic condition when pH_i was lowered by altering the NH₄Cl gradient across the cell membrane. Finally, the effects of SA and NH₄Cl on the membrane excitability and basal tension were studied: Under current clamp mode, resting membrane potential (RMP) was −28 ± 2.3 mV in a single cell level and was depolarized by 13 ± 2.4 mV with 2 mM tetraethylammonium (TEA). SA hyperpolarized and NH₄Cl depolarized RMP by 10 ± 1.9 and 16 ± 4.7 mV, respectively. SA-induced hyperpolarization and relaxation of basal tension was significantly inhibited by TEA. These results suggest that SA and NH₄Cl might regulate vascular tone by altering membrane excitability through modulation of [Ca²⁺]_i and Ca²⁺-activated K channels in rabbit basilar artery.     

Ketamine blocks voltage-gated K+ channels and causes membrane depolarization in rat mesenteric artery myocytes

Clinical doses of ketamine typically increase blood pressure, heart rate, and cardiac output. However, the precise mechanism by which ketamine produces these cardiovascular effects remains unclear. The voltage-gated K⁺ (K_V) channel is the major regulator of resting membrane potential (E _m) and vascular tone in many arteries. Therefore, we sought to evaluate the effects of ketamine on K_V currents using the standard whole-cell patch clamp recordings in single myocytes, enzymatically dispersed from rat mesenteric arteries. Ketamine [(±)-racemic mixture] inhibited K_V currents reversibly and concentration dependently with a K _d of 566.7 ± 32.3 μM and Hill coefficient of 0.75 ± 0.03. The inhibition of K_V currents by ketamine was voltage independent, and the time courses of channel activation and inactivation were little affected. The effects of ketamine on steady-state activation and inactivation curves were also minimal. Use-dependent inhibition was not observed either. S(+)-ketamine inhibited K_V currents with similar potency and efficacy as the racemic mixture. The average resting E _m in rat mesenteric artery myocytes was −44.1 ± 4.2 mV, and both racemic and S(+)-ketamine induced depolarization of E _m (15.8 ± 3.6 and 24.3 ± 5.0 mV at 100 μM, respectively). We conclude that ketamine induces E _m depolarization in vascular myocytes by blocking K_V channels in a state-independent manner, which may contribute to the increased vascular tone and blood pressure produced by this drug under a clinical setting.     

Whole-cell conductive properties of rat pancreatic acini

Acetylcholine-controlled exocrine secretion by pancreatic acini has been explained by two hypotheses. One suggests that NaCl secretion occurs by secondary active secretion as has been originally described for the rectal gland of Squalus acanthias. The other is based on a “push-pull” model whereby Cl⁻ is extruded luminally and sequentially taken up basolaterally. In the former model Cl⁻ uptake is coupled to Na⁺ and basolateral K⁺ conductances play a crucial role, in the latter model, Na⁺ uptake supposedly occurs via basolateral non-selective cation channels. The present whole-cell patch-clamp studies were designed to further explore the conductive properties of rat pancreatic acini. Pilot studies in approximately 300 cells revealed that viable cells usually had a membrane voltage (V _m) more hyperpolarized than −30 mV. In all further studies V _m had to meet this criterion. Under control conditions V _m was −49 ± 1 mV (n = 149). The fractional K⁺ conductance (f _K) was 0.13 ± 0.1 (n = 49). Carbachol (CCH, 0.5 μmol/l) depolarized to −19 ± 1.1 mV (n = 63) and increased the membrane conductance (G _m) by a factor of 2–3. In the seeming absence of Na⁺ [replacement by N-methyl-D-glucamine (NMDG⁺)] V _m hyperpolarized slowly to −59 ± 2 mV (n = 90) and CCH still induced depolarizations to −24 ± 2 mV (n = 34). The hyperpolarization induced by NMDG⁺ was accompanied by a fall in cytosolic pH by 0.4 units, and a very slow and slight increase in cytosolic Ca²⁺. f _K increased to 0.34. The effect of NMDG⁺ on V _m was mimicked by the acidifying agents propionate and acetate (10 mmol/l) added to the bath. The present study suggests that f _K makes a substantial contribution to G _m under control conditions. The NMDG⁺ experiments indicate that the non- selective cation conductance contributes little to V _m in the presence of CCH. Hence the present data in rat pancreatic acinar cells do not support the push-pull model. Received: 8 November 1995/Received after revision: 18 December 1995/Accepted: 3 January 1996     

Effects of antidiuretic hormone,parathyroid hormone and glucagon on transepithelial voltage and resistance of the cortical and medullary thick ascending limb of Henle's loop of the mouse nephron

The effect of antidiuretic hormone (arginine vasopressin, AVP, 10⁻¹⁰mol.l⁻¹), parathyroid hormone (PTH, 10⁻⁸ mol.l⁻⁸) and glucagon (10⁻⁸ mol.l⁻¹) on the transepithelial potential difference (PD_te) and the transepithelial resistance (R_te) were tested in in vitro perfused cortical (cTAL) and medullary (mTAL) thick ascending limbs of Henle's loop of the mouse nephron. When compared with mTAL segments (PD_te: 8.5±0.4 mV,n=16), cTAL segments displayed a high PD_te of 15.7±0.9 mV (n=11) at the beginning of perfusion experiments which reached a value of 9.4±0.6 mV (n =11) after 38±4 min perfusion. Simultaneously R_te increased significantly from 24±3 to 28±1 Ω cm² (n=11). When PTH, AVP or glucagon were added to the bath solution, PD_te increased with PTH from 10.3±0.8 to 15.2±0.8 mV (n=13), with AVP from 10.2±0.5 to 15.0±0.7 mV (n=24) and with glucagon from 11.3±1.9 to 15.3±2.1 mV (n=8). At the same time R_te decreased from 30±3 to 23±2 Ω cm², from 28±1 to 23±1 Ω cm² and from 23±2 to 18±2 Ω cm², respectively. In mTAL segments, AVP and glucagon increased PD_te from 8.4+0.5 to 13.5±0.9 mV (n=11) and from 8.8±0.6 to 12.8±0.6 mV (n=8) respectively, while R_te decreased significantly from 23±1 to 20±1 Ω cm² and from 27±3 to 21±3 Ω cm². PTH, on the other hand, had no effect on PD_te and R_te. Since the response to PTH appeared to be specific to cTAL segments, paired experiments were performed, in which AVP or glucagon were successively tested with PTH on cTAL and mTAL segments, to ascertain the specificity of the hormonal response. In cTAL segments, PTH and AVP increased the equivalent short-circuit current (I_sc=PD_te/R_te) by 82% and 86% respectively, while PTH and glucagon, in another series, increased I_sc by 95% and 81% respectively. In mTAL segments, I_sc was increased in the presence of AVP and glucagon by 88%, and 93% respectively, whereas PTH had no effect. These results indicate that Nacl reabsorption in cTAL segments is stimulated by AVP, PTH and glucagon and in mTAL segments by AVP and glucagon. The amplitude of the response to the hormones is similar in the two segments. The residual stimulation in cTAL segments, however, persists longer than in mTAL segments.     

The contribution of haemoglobin mass to increases in cycling performance induced by simulated LHTL

We sought to determine whether improved cycling performance following ‘Live High-Train Low’ (LHTL) occurs if increases in haemoglobin mass (Hb_mass) are prevented via periodic phlebotomy during hypoxic exposure. Eleven, highly trained, female cyclists completed 26 nights of simulated LHTL (16 h day⁻¹, 3000 m). Hb_mass was determined in quadruplicate before LHTL and in duplicate weekly thereafter. After 14 nights, cyclists were pair-matched, based on their Hb_mass response (ΔHb_mass) from baseline, to form a response group (Response, n = 5) in which Hb_mass was free to adapt, and a Clamp group (Clamp, n = 6) in which ΔHb_mass was negated via weekly phlebotomy. All cyclists were blinded to the blood volume removed. Cycling performance was assessed in duplicate before and after LHTL using a maximal 4-min effort (MMP_4min) followed by a ride time to exhaustion test at peak power output (T _lim). VO_2peak was established during the MMP_4min. Following LHTL, Hb_mass increased in Response (mean ± SD, 5.5 ± 2.9%). Due to repeated phlebotomy, there was no ΔHb_mass in Clamp (−0.4 ± 0.6%). VO_2peak increased in Response (3.5 ± 2.3%) but not in Clamp (0.3 ± 2.6%). MMP_4min improved in both the groups (Response 4.5 ± 1.1%, Clamp 3.6 ± 1.4%) and was not different between groups (p = 0.58). T _lim increased only in Response, with Clamp substantially worse than Response (−37.6%; 90% CL −58.9 to −5.0, p = 0.07). Our novel findings, showing an ~4% increase in MMP_4min despite blocking an ~5% increase in Hb_mass, suggest that accelerated erythropoiesis is not the sole mechanism by which LHTL improves performance. However, increases in Hb_mass appear to influence the aerobic contribution to high-intensity exercise which may be important for subsequent high-intensity efforts.     

Stroke volume equation for impedance cardiography

The study's goal was to determine if cardiac output (CO), obtained by impedance cardiography (ICG), would be improved by a new equation N, implementing a square root transformation for dZ/dt_max/Z₀, and a variable magnitude, mass-based volume conductor V_c. Pulmonary artery catheterisation was performed on 106 cardiac surgery patients pre-operatively. Post-operatively, thermodilution cardiac output (TDCO) was simultaneously compared with ICG CO. dZ/dt_max/Z₀ and Z₀ were obtained from a proprietary bioimpedance device. The impedance variables, in addition to left ventricular ejection time T_LVE and patient height and weight, were input using four stroke volume (SV) equations: Kubicek (K), Sramek (S), Sramek-Bernstein (SB), and a new equation N. CO was calculated as SV × heart rate. Data are presented as mean ± SD. One way repeated measures of ANOVA followed by the Tukey test were used for inter-group comparisons. Bland-Altman methods were used to assess bias, precision and limits of agreement. P<0.05 was considered statistically significant. CO implementing N (6.06±1.48 l min⁻¹) was not different from TDCO (5.97±1.41 l min⁻¹). By contrast, CO calculated using K (3.70±1.53 l min⁻¹), S (4.16±1.83 l min⁻¹) and SB (4.37±1.82 l min⁻¹) was significantly less than TDCO. Bland-Altman analysis showed poor agreement between TDCO and K, S and SB, but not between TDCO and N. Compared with TDCO, equation N, using a square-root transformation for dZ/dt_max/Z₀, and a mass-based V_C was superior to existing transthoracic impedance techniques for SV and CO determination.     

Indirect measures of human vagal withdrawal during head-up tilt with and without a respiratory acidosis

Human ECG records were analyzed during supine (SUP) rest and whole body 80° head-up tilt (HUT), with a respiratory acidosis (5%CO₂) and breathing room air (RA). HUT increased heart rate in both conditions (RA_SUP 60 ± 13 vs. RA_HUT 79 ± 16; 5%CO_2SUP 63 ± 12 vs. 5%CO_2HUT 79 ± 14 beats min⁻¹) and decreased mean R–R interval, with no changes in the R–R interval standard deviation. When corrected for changes in frequency spectrum total power (NU), the high frequency (0.15–0.4 Hz) component (HF_NU) of heart rate variability decreased (RA_SUP 44.01 ± 21.57 vs. RA_HUT 24.05 ± 13.09; 5%CO_2SUP 69.23 ± 15.37 vs. 5%CO_2HUT 47.64 ± 21.11) without accompanying changes in the low frequency (0.04–0.15 Hz) component (LF_NU) (RA_SUP 52.36 ± 21.93 vs. RA_HUT 66.58 ± 19.49; 5%CO_2SUP 22.97 ± 11.54 vs. 5%CO_2HUT 40.45 ± 21.41). Positive linear relations between the tilt-induced changes (Δ) in HF_NU and R–R interval were recorded for RA (ΔHF_NU = 0.0787(ΔR−R) − 11.3, R ² = 0.79, P < 0.05), and for 5%CO₂ (ΔHF_NU = 0.0334(ΔR−R) + 1.1, R ² = 0.82, P < 0.05). The decreased HF component suggested withdrawal of vagal activity during HUT. For both RA and 5%CO₂, the positive linear relations between ΔHF_NU and ΔR−R suggested that the greater the increase in heart rate with HUT, the greater the vagal withdrawal. However, a reduced range of ΔHF during HUT with respiratory acidosis suggested vagal withdrawal was lower with a respiratory acidosis.     

Effect of stretch on calcium channel currents recorded from the antral circular myocytes of guinea-pig stomach

The effect of membrane stretch on voltage-activated Ba²⁺ current (I _Ba) was studied in antral circular myocytes of guinea-pig using the whole- cell patch-clamp technique. The changes in cell volume were elicited by superfusing the myocytes with anisosmotic solutions. Hyposmotic superfusate (202 mosmol/l) induced cell swelling and increased peak values of I _Ba at 0 mV (from −406.6 ± 45.5 pA to −547.5 ± 65.6 pA, mean ± SEM, n = 8) and hyperosmotic superfusate (350 mosmol/l) induced cell shrinkage and decreased peak values of I _Ba at 0 mV (to −269.5 ± 39.1 pA, n = 8). Such changes were reversible and the extent of change was dependent on the osmolarity of superfusate. The values of normalized I _Ba at 0 mV were 1.43 ± 0.04, 1.30 ± 0.06, 1.23 ± 0.04, 1.19 ± 0.04, 1 and 0.68 ± 0.06 at 202, 220, 245, 267, 290 and 350 mosmol/l, respectively (n = 8). I _Ba was almost completely blocked by nicardipine (5 μM) under hyposmotic conditions. The values of steady-state half-inactivation voltage (−37.7 ± 3.3 and −36.5 ± 2.6 mV, under control and hyposmotic conditions, respectively) or the half-activation voltage (−13.6 ± 2.3 and −13.9 ± 1.9 mV) of I _Ba were not significantly changed (P > 0.05, n = 6). Cell membrane capacitance was slightly increased from 50.00 ± 2.86 pF to 50.22 ± 2.82 pF by a hyposmotic superfusate (P < 0.05, n = 6). It is suggested that cell swelling increases voltage-operated L-type calcium channel current and that such a property is related to the response of gastric smooth muscle to mechanical stimuli. Received: 14 November 1995/Received after revision and accepted: 8 January 1996     

Thermodynamic parameters of binding of mouse 2G3 antitheophylline monoclonal antibodies to theophylline

The binding of 2G3 mouse antitheophylline monoclonal antibodies (affinity constant K_aff=2×10¹⁰ liter/mol) to theophylline is studied using isothermal titration microcalorimetry. Thermodynamic parameters of the binding are: enthalpy change ΔH=−0.23 kcal/mol; Gibbs free energy change ΔG=−16.42 kcal/mol; entropy change ΔS=0.054 kcal/(mol×K). Regression analysis shows a two-site kinetic binding model. A great contribution of entropy component to the free energy change of 2G3 antibody binding to theophylline is indicative of an entropy-dependent process. The entropy-dependent nature of the binding presumably determines the binding kinetics. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 124, No. 11, pp. 570–573, November, 1997     

Optimal power-to-mass ratios when predicting flat and hill-climbing time-trial cycling

The purpose of this article was to establish whether previously reported oxygen-to-mass ratios, used to predict flat and hill-climbing cycling performance, extend to similar power-to-mass ratios incorporating other, often quick and convenient measures of power output recorded in the laboratory [maximum aerobic power (W _MAP), power output at ventilatory threshold (W _VT) and average power output (W _AVG) maintained during a 1 h performance test]. A proportional allometric model was used to predict the optimal power-to-mass ratios associated with cycling speeds during flat and hill-climbing cycling. The optimal models predicting flat time-trial cycling speeds were found to be (W _MAP m ^−0.48)^0.54, (W _VT m ^−0.48)^0.46 and (W _AVG m ^−0.34)^0.58 that explained 69.3, 59.1 and 96.3% of the variance in cycling speeds, respectively. Cross-validation results suggest that, in conjunction with body mass, W _MAP can provide an accurate and independent prediction of time-trial cycling, explaining 94.6% of the variance in cycling speeds with the standard deviation about the regression line, s=0.686 km h⁻¹. Based on these models, there is evidence to support that previously reported -to-mass ratios associated with flat cycling speed extend to other laboratory-recorded measures of power output (i.e. Wm ^−0.32). However, the power-function exponents (0.54, 0.46 and 0.58) would appear to conflict with the assumption that the cyclists’ speeds should be proportional to the cube root (0.33) of power demand/expended, a finding that could be explained by other confounding variables such as bicycle geometry, tractional resistance and/or the presence of a tailwind. The models predicting 6 and 12% hill-climbing cycling speeds were found to be proportional to (W _MAP m ^−0.91)^0.66, revealing a mass exponent, 0.91, that also supports previous research.     

Oxygen uptake kinetics during severe intensity running and cycling

The purpose of this study was to investigate the effect of exercise mode on the characteristics of the oxygen uptake ( V̇O₂) response to exercise within the severe intensity domain. Twelve participants each performed a treadmill running test and a cycle ergometer test to fatigue at intensities selected to elicit a mode-specific V̇O₂max and to cause fatigue in ~5 min. The tests were at 234 (30) m·min⁻¹ and 251 (59) W, and times to fatigue were 297 (15) s and 298 (14) s, respectively. The overall rapidity of the V̇O₂response was influenced by exercise mode [ V̇O₂max was achieved after 115 (20) s in running versus 207 (36) s in cycling; p<0.01]. V̇O₂ responses were fit to a three-phase exponential model. The time constant of the primary phase was faster in treadmill tests than in cycle ergometer tests [14 (6) s versus 25 (4) s; p<0.01], and the amplitude of the primary phase was greater in running than in cycling when it was expressed in absolute terms [2327 (393) ml·min⁻¹ versus 2036 (301) ml·min⁻¹; p=0.02] but not when it was expressed as a percentage of the total increase in V̇O₂ [86 (6)% versus 82 (6)%; p=0.09]. When quantified as the difference between the end-exercise V̇O₂ and the V̇O₂ at 2 min, the amplitude of the slow component was ~40% smaller in running [177 (92) ml·min⁻¹ versus 299 (153) ml min⁻¹; p=0.03]. It is concluded that exercise modality affects the characteristics of the V̇O₂ response at equivalent intensities in the severe domain.     

Non-invasive imaging of optical parameters of biological tissues

The normalised back-scattered intensity (NBI) profiles at various locations on the forearms of ten human subjects were obtained by moving the multi-probe of a laser reflectometer. The statistical analysis of the NBI data showed that the variation in the NBI was significantly higher at the ulnar region compared with that at other regions. For determination of the scattering (μ _s ) and absorption (μ _a ) coefficients and the anisotropy parameter g at each location on the forearm, these profiles were matched with the NBI profiles simulated by a Monte Carlo procedure (χ _0.99 ² ). For the reconstruction of images of variation of these parameters, the averaged values ofμ _a ,μ _s and g at all locations on the forearms of the subjects were determined. The absorption coefficient had a minimum (1.92 cm⁻¹) and maximum (2.21 cm⁻¹) at the wrist and the lateral region of the forearm, respectively. The scattering coefficient had a maximum (194 cm⁻¹) at the medial side and near the elbow, and a minimum (186 cm⁻¹) at the lateral side of the forearm. Similar changes in the anisotropy parameter were also observed. By interpolation of the data of each parameter on a 100×100 image matrix and after median filtering, colour-coded images of the variation in the optical parameters were constructed. These images could be useful for diagnostic and therapeutic applications of lasers.     

Volume-sensitive chloride currents in primary cultures of human fetal vas deferens epithelial cells

Using the patch-clamp technique, we have identified a large, outwardly rectifying, Cl⁻-selective whole-cell current in primary cultures of human vas deferens epithelial cells. Whole-cell currents were time- and voltage-dependent and displayed inactivation following depolarising pulses ≥ 60 mV. Currents were equally permeable to bromide (P _Br/P _Cl = 1.05 ± 0.04), iodide (P _I/P _Cl = 1.06 ± 0.07) and Cl⁻, but significantly less permeable to gluconate (P _Gluc/P _Cl = 0.23 ± 0.03). Currents spontaneously increased with time after establishing a whole-cell recording, but could be inhibited by exposure to a hypertonic bath solution which reduced inward currents by 68 ± 4%. Subsequent exposure of the cells to a hypotonic bath solution led to a 418 ± 110% increase in inward current, indicating that these currents are regulated by osmolarity. 4,4′-Diisothiocyanatostilbene-2,2′-disulphonic acid (100 μM) produced a rapid and reversible voltage-dependent block (60 ± 5% and 10 ± 7% inhibition of current, measured at ± 60 mV, respectively). Dideoxyforskolin (50 μM) also reduced the volume-sensitive Cl⁻ current, but with a much slower time course, by 41 ± 13% and 32 ± 16% (measured at ± 60 mV, respectively). Tamoxifen (10 μM) had no effect on the whole-cell Cl⁻ current. These results suggest that vas deferens epithelial cells possess a volume-sensitive Cl⁻ conductance which has biophysical and pharmacological properties broadly similar to volume-sensitive Cl⁻ currents previously described in a variety of cell types. Received: 25 January/Accepted: 25 April 1996     

A stretch-activated K+ channel in the basolateral membrane ofXenopus kidney proximal tubule cells

The present study examined whether a basolateral potassium ion (K⁺) channel is activated by membrane-stretching in the cell-attached patch. A K⁺ channel of conductance of 27.5 pS was most commonly observed in the basolateral membrane ofXenopus kidney proximal tubule cells. Channel activity increased with hyperpolarizing membrane potentials [at more positive pipette potentials (V _p)]. Open probability (P _o) was 0.03, 0.13, and 0.21 atV _p values of 0, 40, and 80 mV, respectively. Barium (0.1 mM) in the pipette reducedP _o by 79% at aV _p of 40 mV. Application of negative hydraulic pressure (−16 to −32 cm H₂O) to the pipette markedly activated outward currents (fromP _o=0.01 to 0.75) at aV _p of −80 mV, but not inward currents at aV _p of 80 mV. The size of the activated outward currents (from cell to pipette) did not change by replacing chloride with gluconate in the pipette. These results indicate that a stretch-activated K⁺ channel exists in the basolateral membrane of proximal tubule cells. It may play an important role as a K⁺ exit pathway when the cell membrane is stretched (for example, by cell swelling).