Force–velocity and unloaded shortening velocity during graded potassium contractures in frog skeletal muscle fibres

Steady-state conditions of contraction, at maximal and submaximal forces, were produced in intact single muscle fibres, from Rana esculenta, using full tetani and graded K⁺-contractures. The uniformity in radial direction, of spreading of activation produced in K⁺-contractures, was checked in relation to the fibre diameters. The absolute isometric force was similar in tetani and maximal contractures, for fibres with diameters between 40 and 60 m, but not for fibres with diameters greater than about 70 m in which contracture force never reached tetanic force. The force–[K⁺]_o relation was similar for fibres with diameters between 40 and 60 m, but it was right shifted and it had a minor slope for fibres with diameters greater than 65–70 m. This suggests that only in the small diameter fibres (40–60 m) the activation does not fail to penetrate uniformly from the surface towards the fibre core. For fibres selected in the diameter range between 40 and 60 m, force–velocity relations and unloaded shortening velocities were determined in tetani and maximal and submaximal contractures. Data were obtained across a force range of 0.3 to 1 P ₀ (tetanic plateau force). Controlled velocity method was used to obtain force–velocity relations, and slack test to determine the unloaded shortening velocity (V _U). The values of the parameters characterising the force–velocity relation (V ₀ and a/P ₀) and V _U as determined by the slack test did not differ significantly in tetani and contractures, independent of the activation level or absolute force developed by the fibre. These results show that, at least within the range of forces tested, crossbridge kinetics is independent of the number of cycling crossbridges, in agreement with the prediction of the recruitment model of myofilament activation.     

Differential effects of halothane on adult and juvenile sodium channels in human muscle

Myoballs were cultured from biopsies of adult human skeletal muscle. Transient currents through the sodium channels were elicited by depolarizing a myoball membrane with the whole-cell patch-clamp technique. The properties of the sodium channels were determined from the Hodgkin-Huxley parameters (I _{Na max}, _m, _h,h -curve) derived from these transients. Halothane, when applied at 3.4 mmol/l (15 kPa), blocked about 50% of the current through the adult, TTX-sensitive sodium channels but had little effect on the current through the juvenile, TTX-insensitive sodium channels. At >12 mmol/l, halothane blocked both channel types completely. The time constants of activation and inactivation were decreased in the presence of 3.4 mmol/l halothane but not enough to account for the decrease of the current amplitude. Halothane shifted the h-curves of both channel types towards more negative potentials by an amount that was roughly proportional to its concentration. Myoballs from a man susceptible to malignant hyperthermia (MH) gave the same results as the controls indicating that the halothane effects on the action potential of MH-susceptible muscle are not mediated by a specific effect on the sodium channels.     

Modification of electrophysiological and pharmacological properties of K channels in neuroblastoma cells induced by the oxidant chloramine-T

The effects of chloramine-T (CL-T) on voltage-dependent potassium channels in neuroblastoma cells were analysed using the whole-cell current recording technique. CL-T irreversibly decreased the peak whole — cell K current, considerably slowed its inactivation and shifted its activation-voltage curve towards positive voltages by 6 mV. Under control conditions, the inactivation of the whole-cell K current could be described by the sum of two exponentials, F and S, whose time constants at +50 mV were _F=1.00±0.15 s and _S=5.72±0.47 s respectively. After CL-T, it could be described by the sum of two (S₁ and S₂) or three (F, S₁ and S₂) exponentials whose time constants at +50 mV were: _F=0.81±0.22 s, _S1=6.46±0.60 s and _S2=48.56±3.64 s. Under control conditions, F and S inactivating components of the whole-cell K current were blocked by 4-aminopyridine, with a Hill coefficient of 1 and apparent dissociation constants of 0.04 and 0.7 mM respectively. After CL-T, both S₁ and S₂ components were equally blocked by 4-aminopyridine with a Hill coefficient of 0.25, being reduced to 64% of their control values by 10 mM. CL-T is known to slow the inactivation of sodium channels and to oxidize sulphydryl amino acids and unsaturated lipids. It is concluded that the inactivation gates of voltage-dependent sodium and potassium channels are either constituted of the same amino acid residues or are controlled by unsaturated lipids surrounding or bound to the channel proteins.     

Does critical swimming velocity represent exercise intensity at maximal lactate steady state?

Summary The purpose of this investigation was to determine whether the critical swimming velocity ( _crit), which is employed in competitive swimming, corresponds to the exercise intensity at maximal lactate steady state. _crit is defined as the swimming velocity which could theoretically be maintained forever without exhaustion and expression as the slope of a regression line between swimming distances covered and the corresponding times. A total of eight swimmers were instructed to swim two different distances (200 m and 400 m) at maximal effort and the time taken to swim each distance was measured. In the present study, _crit is calculated as the slope of the line connecting the two times required to swim 200 m and 400 m. vcrit determined by this new simple method was correlated significantly with swimming velocity at 4 mmol · 1^–1 of blood lactate concentration (r = 0.914,P < 0.01) and mean velocity in the 400m freestyle (r = 0.977,P < 0.01). In the maximal lactate steady-state test, the subjects were instructed to swim 1600 m (4 x 400 m) freestyle at three constant velocities (98010, 100% and 102070 of _crit). At 100% _crit blood lactate concentration showed a steady-state level of approximately 3.2 mmol · 1^– from the first to the third stage and at 98% of _crit lactate concentration had a tendency to decrease significantly at the fourth stage. On the other hand, at 102% of _crit, blood lactate concentration increased progressively and those of the third and fourth stages were significantly higher than those at 100% of _crit (P<0.05). These data suggest that _crit, which can be calculated by performing two timed, maximal effort swimming tests, may correspond to the exercise intensity at maximal lactate steady state.     

Recombinant human interleukin 1β and tumor necrosis factor affect glycosylation of serumα 1-acid glycoprotein in rats

Serum concentration and glycosylation of rat ₁,-acid glycoprotein ( ₁-AGP) were evaluated after the in vivo administration of recombinant human interleukin-1 (rhIL-1) and tumor necrosis factor (rhTNF-), alone or associated. The effect of LPS and turpentine was also studied. In all models, serum ₁-AGP concentrations were increased and glycosylation was altered. The ₁-AGP levels reached 1.8 g/liter with cytokines alone, 2.1 g/liter with cytokines associated or LPS, and 3.4 g/liter with turpentine. Analysis by concanavalin A (Con A) affinoimmunoelectrophoresis (CAIE) revealed that the relative proportion of Con A unreactive form always decreased whatever the inducing agent. On the other hand, the resulting effect on the concentrations cf Con A unreactive ₁-AGP concentrations was an increase with cytokines alone or LPS and a decrease with cytokines associated or turpentine. These results suggest a dissociation between the alteration in the level of ₁-AGP synthesis and in the pattern of its glycosylation in the various inflammatory models.     

Voltage-dependent noradrenergic modulation of ω-conotoxin-sensitive Ca2+ channels in human neuroblastoma IMR32 cells

High-threshold (HVA) Ca²⁺ channels of human neuroblastoma IMR32 cells were effectively inhibited by noradrenaline. At potentials between –20 mV and +10 mV, micromolar concentrations of noradrenaline induced a 50%–70% depression of HVA Ba²⁺ currents and a prolongation of their activation kinetics. Both effects were relieved at more positive voltages or by applying strong conditioning pre-pulses (facilitation). Facilitation restored the rapid activation of HVA channels and recruited about 80% of the noradrenaline-inhibited channels at rest. Re-inhibition of Ca²⁺ channels after facilitation was slow ( _r 36–45 ms) and voltage-independent between –30 mV and –90 mV. The inhibitory action of noradrenaline was dose-dependent (IC₅₀=84 nM), mediated by ₂-drenergic receptors and selective for -conotoxin-sensitive Ca²⁺ channels, which represent the majority of HVA channels expressed by IMR32 cells. The action of noradrenaline was mimicked by intracellular applications of GTP[S] and prevented by GDP[S] or by pre-incubation with pertussis toxin. The time course of noradrenaline inhibition measured during fast application (onset) and wash-out (offset) of the drug were independent of saturating agonist concentrations (10–50 M) and developed with mean time constants of 0.56 s ( _on) and 3.6 s ( _off) respectively. The data could be simulated by a kinetic model in which a G protein is assumed to modify directly the voltage-dependent gating of Ca²⁺ channels. Noradrenaline-modified channels are mostly inhibited at rest and can be recruited in a steep voltage-dependent manner with increasing voltages.     

Ventilatory responses to exercise and carbon dioxide in elderly and younger humans

Summary The present investigation examined the relationship between CO₂ sensitivity [at rest (S _R) and during exercise (S _E)] and the ventilatory response to exercise in ten elderly (61–79 years) and ten younger (17–26 years) subjects. The gradient of the relationship between minute ventilation and CO₂ production ( _E/ CO₂) of the elderly subjects was greater than that of the younger subjects [mean (SEM); 32.8 (1.6) vs 27.3 (0.4); P<0.01]. At rest, S _R was lower for the elderly than for the younger group [10.77 (1.72) vs 16.95 (2.13) 1 · min^–1 · kPa^–1; 1.44 (0.23) vs 2.26 (0.28) 1 · min^–1 · mmHg^–1; P<0.05], but S _E was not significantly different between the two groups [17.85 (2.49) vs 19.17 (1.62) l · min^–1 · kPa^–1; 2.38 (0.33) vs 2.56 (0.21) 1 · min^–1 · mmHg^–1]. There were significant correlations between both S _R and S _E, and _E/ CO₂ (P<0.05; P<0.001) for the younger group, bot none for the elderly. The absence of a correlation for the elderly supports the suggestion that _E/ CO₂ is not an appropriate index of the ventilatory response to exercise for elderly humans.     

Effects of the reactive oxygen species hypochlorous acid and hydrogen peroxide on force production and calcium sensitivity of rat cardiac myofilaments

Neutrophil activation occurs after myocardial infarction/ischaemia. They produce the reactive oxygen species (ROS) hypochlorous acid (HOCl) and hydrogen peroxide (H₂O₂) which could contribute to contractile dysfunction upon reperfusion. The myofilaments of skinned rat cardiac muscle were exposed to ROS in various states of activation. Isometric force was measured at controlled degrees of activation. A single application of 10 M HOCl for 1 min increased log [Ca²⁺] for half-maximal activation (log K _1/2) from 5.23 to 5.32, initial maximum Ca-activated force (F _{Ca, max}) was reduced by 18.8±5.8% and resting tension increased to 15.4±8.0% of F _{Ca, max}. At 50 M, a 1-min exposure to HOCl produced a greater increase in Ca-sensitivity (log K _1/2 increased from 5.23 to 5.47), a greater reduction in F_{Ca, max} (falling by 42.3±23.2%) and a greater increase in resting tension (to 25±10.7% of F _{Ca, max}). The nature of the resting tension rise was examined by reducing pH before and during exposure to HOCl; the results are consistent with rigor-like cross-bridges being involved. H₂O₂ was without effect on the myofilaments at physiologically relevant (< 10 M) concentrations. These results suggest that ROS associated with inflammation could contribute to post-ischaemic myocardial dysfunction.     

Structure and mechanics of growing arterial microvessels from hypertrophied urinary bladder in the rat

Rat bladder hypertrophy, induced by a partial ligation of the urethra, was used to study the accompanying changes of microvascular smooth muscle mechanics, pharmacology and morphology. A segment of a microarterial vessel to the bladder was taken from a defined anatomical location and studied in a wire myograph in vitro at the length for maximal isometric force development (L _max). After 10 days of ligation, bladder hypertrophy resulted in a microvascular growth response compared to non-operated controls which was characterized by (i) an increase of the calculated diameter at L _max from 134±5 m to 222±19 m; (ii) an increase of the media thickness from 22.4±1.9 m to 32.2±3.0 m; (iii) an increase of the active tension from 1.42±0.28 mN/mm to 3.06±0.33 mN/mm; (iv) no change of the wall/lumen ratio (from 0.83±0.10 to 0.79±0.15). Normalized length/force relations (active, passive and total) did not differ significantly between microarteries from control and hypertrophic bladders. Microvascular smooth muscle growth was also associated with a decreased sensitivity to K⁺-induced depolarization and an increased sensitivity to ₁-adrenergic stimulation. No differences were noted regarding the Ca²⁺ sensitivity of force during K⁺-induced depolarization. The results suggest that microvascular growth (1) is immediately and positively influenced by the organ growth; (2) results in a functional resetting of the microvascular segments towards larger diameters without gross morphological or mechanical alterations; and (3) is accompanied by pharmacological alterations of the smooth muscle reactivity.     

Multiple calcium channel subtypes in isolated rat chromaffin cells

By using the whole-cell configuration of the patch-clamp technique we have investigated the pharmacological properties of Ca²⁺ channels in short-term cultured rat chromaffin cells. In cells held at a membrane potential of –80 mV, using 10 mM Ba²⁺ as the charge carrier, only high-voltage-activated (HVA) Ca²⁺ channels were found. Ba²⁺ currents (I _Ba) snowed variable sensitivity to dihydropyridine (DHP) Ca²⁺ channel agonists and antagonists. Furnidipine, a novel DHP antagonist, reversibly blocked the current amplitude by 22% and 48%, at 1 M and 10 M respectively, during short (15–50 ms) depolarizing pulses to 0 mV. The L-type Ca²⁺ channel agonist Bay K 8644 (1 M) caused a variable potentiation of HVA currents that could be better appreciated at low rather than at high depolarizing steps. Increase of I _Ba was accompanied by a 20-mV shift in the activation curves for Ca²⁺ channels towards more hyperpolarizing potentials. Application of the conus toxin -conotoxin GVIA (GVIA; 1 M) blocked 31% of I _Ba; blockade was irreversible upon removal of the toxin from the extracellular medium, -Agatoxin IVA (IVA; 100 nM) produced a 15% blockade of I _Ba. -Conotoxin MVIIC (MVIIC; 5 M) produced a 36% blockade of I _Ba; such blockade seems to be related to both GVIA-sensitive (N-type) and GVIA-resistant Ca²⁺ channels. The sequential addition of supramaximal concentrations of furnidipine (10 M), GVIA (1 M), IVA (100 nM) and MVIIC (3 M) produced partial inhibition of I _Ba, which were additive. Our data suggest that the whole cell I _Ba in rat chromaffin cells exhibits at least four components. About 50% of I _Ba is carried by L-type Ca²⁺ channels, 30% by N-type Ca²⁺channels and 15% by P-type Ca²⁺ channels. These figures are close to those found in cat chromaffin cells. However, they differ considerably from those found in bovine chromaffin cells where P-like Ca²⁺channels account for 45% of the current, N-type carry 35% and L-type Ca²⁺ channels are responsible for only 20–25% of the current. These drastic differences might have profound physiological implications for the relative contribution of each channel subtype to the regulation of catecholamine release in different animal species.     

Gating current experiments on frog nodes of Ranvier treated withCentruroides sculpturatus toxins or aconitine

(1) Gating currents were recorded from frog nodes of Ranvier treated either with toxins III or IV from the venom of the scorpionCentruroides sculpturatus or with the alkaloid toxin aconitine. (2) Toxins III or IV fromCentruroides sculpturatus (which drastically reduce the sodium permeabilityP _Na and slightly shift its voltage dependence in the depolarizing direction) caused a small depolarizing shift of the relation between charge (Q _on) and membrane potential (E) without affecting the maximum chargeQ _{on max}. (3) On nodes treated with toxins III or IV fromCentruroides sculpturatus, a depolarizing conditioning pulse (which transiently shifts the descending branch of theI _Na(E) curve by up to 60 mV in the hyperpolarizing direction) shifted the midpoint potential (E_mid) of theQ _on(E) curve by –17 mV and slightly increased the slope of the curve; it also decreasedQ _{on max} markedly but had little effect onQ _on measured with small depolarizing pulses. By contrast, massive treatment with aconitine (which irreversibly shifts sodium activation in the hyperpolarizing direction) irreversibly shifted the midpoint potential of theQ _on(E) curve from –28.5 to –69 mV and significantly increasedQ _on andQ _off measured with small depolarizing pulses; concomitantly, the voltage dependence of the on time constant of the charge movement [_on(E)] was shifted by –44 mV. (4) The sodium currentI _Na was exponential both in nodes treated with toxins III or IV ofCentruroides sculpturatus and subjected to a depolarizing conditioning pulse and in aconitine-treated nodes; in the latter,I _Na started after a delay of 30–40 s. The time constant of the sodium current, _{on Na}, was larger than the time constant of the charge movement, _{on Q}; the ratio _{on Q}/_{on Na} was 0.61 and 0.73 in the experiments withCentruroides sculpturatus toxins and aconitine, respectively. (5) The off time constant of the sodium current (_{off Na}) was slightly increased in nodes treated withCentruroides sculpturatus toxins and subjected to a depolarizing conditioning pulse, whereas it was markedly increased in aconitine-treated nodes. With the former treatment, the off time constant of the charge movement (_{off Q}) was unaffected but with aconitine treatment it was considerably increased although it remained smaller than _{off Na}. Consequently, the ratio _{off Q}/_{off Na} (which is 1 in untreated nodes) became smaller than one, reaching values as low as 0.58 and 0.44 in the experiments withCentruroides sculpturatus toxins and aconitine, respectively. The small _{off Q}/_{off Na} ratio suggests that the channels remain open for an appreciable time after most of the gating charges have returned to their resting position. (6) The results obtained with aconitine resemble the findings on batrachotoxin-treated nodes (Dubois and Schneider 1985), except that in the latter the time constants _{on Na} and _{off Na} of the sodium current are smaller than the corresponding time constants _{on Q} and _{off Q} of the charge movement.     

Inositol 1,4,5-trisphosphate mediates adrenaline activation of K+ conductance in mouse peritoneal macrophages

In mouse peritoneal macrophages, ₁-adrenoceptor stimulation evokes a Ca²⁺-dependent K⁺ current [I ₀(Adr)] [Hara et al. (1991) Pflügers Arch 419:371–379]. The roles of D-myo-inositol 1,4,5-trisphosphate (InsP ₃) and a GTP-binding protein (G protein) in I ₀(Adr) were investigated with tight-seal whole-cell recordings and fura-2 fluorescence measurements. Intracellular injection of lnsP ₃ (5–50 M) evoked transient outward currents [I ₀(InsP ₃)] with or without damped oscillations in membrane currents at -40 mV. Dialysis with 0.2 mM guanosine 5-[3-thio]triphosphate (GTP[S], a poorly hydrolysable GTP analogue) at -40 mV activated oscillatory outward currents or a slowly developing steady current on which such oscillations were superimposed after a delay of 10–90 s. I ₀(InsP ₃) and the GTP[S]-induced current {I ₀(GTP[S])} were accompanied by an increase in conductance. Reversal potentials of both responses closely depended on the extracellular K⁺ concentration. Fura-2 measurements revealed that I ₀(InsP ₃) and I ₀(GTP[S]) result from a rise in intracellular free Ca²⁺ concentration ([Ca²⁺]_i). Removal of extracellular Ca²⁺ did not abolish I ₀(InsP ₃) and I ₀(GTP[S]). Both were blocked by bath-applied charybdotoxin. Intracellular D- myo-inositol 1,3,4,5-tetrakisphosphate (InsP ₄, 50 M) did not evoke any responses, whereas D-myo-inositol 2,4,5-trisphosphate [InsP ₃(2,4,5), 20 M] elicited an outward current at -40 mV. I₀(InsP ₃) was completely blocked by prior dialysis with the InsP ₃ receptor antagonist heparin (5 mg/ml). Inclusion of guanosine 5-[2-thio] diphosphate (GDP[S], 2 mM) or heparin (5 mg/ml) together with GTP[S] in the patch pipette solution completely blocked I ₀(GTP[S]). These results indicate that intracellular injection of InsP ₃ or GTP[S] mimic I ₀(Adr). Furthermore, intracellular dialysis with heparin (3 mg/ ml) or GDP[S] (2 mM) greatly accelerated a run-down of I ₀(Adr). On the other hand, I ₀(Adr) was markedly prolonged in a cell dialysed with GTP[S] (0.2 mM). Therefore, it is concluded that I ₀(Adr) results from stimulation of ₁-adrenoceptor and InsP ₃ formation via a G protein.     

Attachment, invasion, chemotaxis, and proteinase expression of B16-BL6 melanoma cells exhibiting a low metastatic phenotype after exposure to dietary restriction of tyrosine and phenylalanine

We previously reported that low levels of tyrosine (Tyr) and phenylalanine (Phe) alter the metastatic phenotype of B16-BL6 (BL6) murine melanoma and select for tumor cell populations with decreased lung colonizing ability. To more specifically characterize the effects of Tyr and Phe restriction on the malignant phenotype of BL6, we investigated in vitro attachment, invasion, proteinase expression, and chemotaxis of high and low metastatic BL6 variants. High metastatic variant cells were isolated from subcutaneous tumors of mice fed a nutritionally complete diet (ND cells) and low metastatic variant cells were isolated from mice fed a diet restricted in Tyr and Phe (LTP cells). Results indicate that attachment to reconstituted basement membrane (Matrigel) was significantly reduced in LTP cells as compared to ND cells. Attachment to collagen IV, laminin, and fibronectin were similar between the two variants. Invasion through Matrigel and growth factor-reduced Matrigel were significantly decreased in LTP cells as compared to ND cells. Zymography revealed the presence of M _r 92 000 and M _r 72 000 progelatinases, tissue plasminogen activator, and urokinase plasminogen activator in the conditioned medium of both variants; however, there were no differences in activity of these secreted proteinases between the two variants. Growth of the variants on growth factor-reduced Matrigel similarly induced expression of the M _r 92 000 progelatinase. The variants exhibited similar chemotactic responses toward laminin. However, the chemotactic response toward fibronectin by LTP cells was significantly increased. MFR5, a monoclonal antibody which selectively blocks function of the ₅ chain of the ₅ß₁ integrin, VLA-5, decreased the chemotactic response toward fibronectin of ND cells by 37%; the chemotactic response by LTP cells was reduced by 49%. This effect was specific for fibronectin-mediated chemotaxis since the chemotaxis toward laminin and invasion through Matrigel were not altered by the presence of MFR5. The surface expression of VLA-5 was significantly increased in LTP cells as compared to ND cells by flow cytometric analysis. These observations suggest that limitation of Tyr and Phe either directly modifies BL6 or selects for subpopulations with altered in vitro invasion, chemotaxis, and integrin expression.     

Selective inhibition of group II phospholipase A2 by quercetin

The influence of quercetin, chlorpromazine, aristolochic acid, and indomethacin on group I phospholipase A₂ (PLA₂) from porcine pancreas and on group II PLA₂ fromVipera russelli was compared. Quercetin and chlorpromazine were found to inhibit PLA₂ activity in lower concentrations (< 100M), while aristolochic acid and indomethacin were inhibitory only in higher concentrations (> 100M). The order of potency againstVipera PLA₂ was: quercetin >chlorpromazin aristolochic acid > indomethacin, while the order of potency against pancreatic PLA₂ was: chlorpromazine > aristolochic acid > indomethacin> quercetin. Thus, quercetin was a potent inhibitor towards group II PLA₂ (IC₅₀=2M), but a very weak inhibitor against group I PLA₂, with maximum 30% inhibition. Aristolochic acid and indomethacin were three to four times more potent towards group II PLA₂ than towards group I PLA₂, while chlorpromazine was equally potent towards the two PLA₂ types. Quercetin and chlorpromazine were also tested against two PLA₂ fractions purified from the plasma of septic shock patients; chlorpromazine was then equally potent towards the two PLA₂ fractions, whereas quercetin was a potent inhibitor of only one of the two PLA₂ fractions (IC₅₀=4M). Together, these results indicate that (1) different PLA₂ inhibitors have different potency depending on which type of PLA₂ they are used against, (2) quercetin selectively inhibits group II PLA₂ and may therefore be used to discriminate between different PLA₂ forms in biological materials, and (3) both PLA₂ of group I and group II are present in septic shock plasma.     

Regulation of resting ionic conductances in frog skeletal muscle

The membrane electrical properties and resting ionic conductances of frog semitendinosus muscle fibres were studied in vitro at 25° C with the two-microelectrode cable technique, in the presence of an activator or inhibitor of protein kinase C (PKC) or in the presence of an activator of adenylate cyclase. The PKC activator, 4-phorbol 12,13-dibutyrate (4-PDB), reduced chloride conductance (G _Cl) at concentrations greater than 1 M and did not affect potassium conductance (G _K). At 150 M, the maximum concentration of 4-PDB tested, G _Cl was reduced by 42%. The inactive phorbol ester 4-phorbol 12,13-dibutyrate did not affect G _Cl or G _K. The inhibitory effect of 4-PDB on G _Cl was prevented by pretreatment of the muscle preparation with the PKC inhibitor staurosporine. The adenylate cyclase activator forskolin (1.5–8 M) significantly increased the G _K of the fibres, without affecting G _Cl. Thus, we conclude that frog skeletal muscle G _Cl, unlike rat muscle G _Cl, is relatively insensitive to activators of PKC. Moreover, in frog muscle, protein kinase A is a likely modulator of G _K, but not G _Cl.     

Point mutations in IIS4 alter activation and inactivation of rat brain IIA Na channels in Xenopus oocyte macropatches

Macroscopic currents of wild-type rat brain IIA (RBIIA) and mutant Na channels were recorded in excised patches from Xenopus oocytes. A charge deletion (K859Q) and an adjacent conservative mutation (L860F) in the second domain S4 membrane-spanning region differentially altered voltage sensitivity and kinetics. Analysis of voltage dependence was confined to Na currents with fast inactivation kinetics, although RBIIA and K859Q (but not L860F) also showed proportional shifts between at least two gating modes, rendering currents with fast or slow inactivation kinetics, respectively. Compared to RBIIA, the midpoint of the activation curve was shifted in both K859Q and L860F by 22 mV to more positive potentials, yet this shift was not associated with a corresponding change in the voltage dependence of time constants for activation ( _a) or inactivation ( _h1, _h2). L860F showed faster activation time constants _a than RBIIA, while K859Q was slower for both the activation ( _a) and the inactivation components ( _h1). Similarly, the steady-state inactivation curve of L860F but not K859Q shifted by 9 mV in the hyperpolarizing direction. Thus, the fourth charge in the IIS4 transmembrane segment exerts control over voltage sensitivity and kinetics of activation and may interact with structure that influence other aspects of channel gating.     

Inhibition of cysteinyl-leukotriene production by azelastine and its biological significance

Azelastine is a phthalazinone derivative with a wide spectrum of pharmacological activities. Actively sensitized guinea pigs were used to examine the broncholytic effect of azelastinein vivo. Furthermore, the influence of azelastine on the production of arachidonic acid (AA) metabolites was investigatedin vitro and compared to the effects of nordihydroguaiaretic acid (NDGA), indomethacin and ketotifen.In vivo, azelastine protected actively sensitized guinea-pigs against ovalbumin-induced bronchospasm with an ID₅₀ of 0.08 mg/kg orally. Ketotifen was similarly active (ID₅₀=0.05 mg/kg). Antigen-induced contraction of isolated tracheal rings of sensitized guinea-pigs was concentration-dependently inhibited by azelastine and NDGA with IC₅₀-values of 94.1 and 34.2 mol/l, respectively. Ketotifen exerted only weak inhibitory activity (18% at 100 mol/l). The arachidonic acid-induced contraction of isolated guinea-pig tracheal rings was also inhibited both by azelastine (IC₅₀=92.6 mol/l) and NDGA (IC₅₀=20.4 mol/l). Ketotifen was inactive on this model. Antigen challenge of chopped lung tissue from sensitized guinea-pigs resulted in the release of cysteinyl-leukotrienes (LT) which were identified by reversed phase high pressure liquid chromatography (HPLC) as LTD₄ and LTE₄. The release of cysteinyl-LT from sensitized guinea-pig lung tissue induced by antigen challenge was concentration-dependently inhibited by azelastine (IC₅₀=35.2 mol/l) and NDGA (IC₅₀=8.4 mol/l) but not by ketotifen and indomethacin. By contrast, indomethacin caused a pronounced augmentation of cysteinyl-LT release. The concentration of indomethacin, which augmented cysteinyl-LT release by 50% was 0.19 mol/l. At the same concentration, indomethacin inhibited the release of 6-keto-PGF₁ and TXB₂ by about 50%. Azelastine negligible influenced 6-keto-PGF₁ and slightly diminished TXB₂ release from the chopped lung tissue after challenge. Its IC₅₀-values were >2 mmol/l and 443 mol/l, respectively. NDGA inhibited the release of 6-keto-PGF₁ and TXB₂ with IC₅₀-values of 47.3 and 38.3 mol/l, respectively. Ketotifen was ineffective in inhibiting the release of cyclo-oxygenase products of AA metabolism. It seems likely that inhibition of release of 5-lipoxygenase-derived products of AA metabolism by azelastine contributes to its antiallergic and antiasthmatic activity.Author for correspondence.     

Intrapulmonary gas mixing and pulmonary gas exchange in artificially ventilated dogs

To investigate the effect of incomplete gas mixing between tidal air and residual gas on pulmonary gas exchange, anaesthetized dogs were ventilated artificially with breathing patterns with different durations of the post-inspiratory apnoea (t _a=0,0.5,1.0 and 2.0 s), where tidal volume, breathing frequency, inspiratory and expiratory flow patterns were kept constant. We determined the alveolar ventilations (V ) of He and SF₆ from the product of end-expiratory lung volume (V _L,E) and specific ventilation (V /V_L,E). V_L,E was determined by the dilution technique and the specific ventilations of the two gases were obtained from their multiple-breath washout. Further, tracer amounts of acetone, ether and enflurane were infused continuously into a peripheral vein and a bolus of a gas mixture of krypton, Freon12 and SF₆ was introduced into the peritoneal cavity. We determined the Excretion (E) and Retention (R) of these six gases according to the multiple-inert-gas-elimination technique (MIGET). V _A increased with increasing t _a, where V _A,He was about 14% larger than V _A,SF6 For both gases, however, the increase in V _A relative to control (V _A for t _a=0) was virtually the same: 9, 11 and 19% (mean values) for t _a=0.5, 1.0 and 2.0 s respectively. For all dogs the E/R curve shifted to larger E values with increasing t_a. E for the most soluble tracer gas (acetone) increased by 11, 21 and 25% for t_a=0.5, 1.0 and 2.0 s respectively. V _A, determined with MIGET from the ventilation/perfusion distribution, increased by almost the same percentages. These results are interpreted to indicate that pulmonary gas exchange is substantially impaired by incomplete intra-acinar gas mixing.     

Proximal tubular stop flow pressure: an index of glomerular capillary pressure?

Central to the assumption that glomerular capillary pressure (P _gc) can be equated with the sum of arterial oncotic pressure ( _art) and the pressure in a blocked proximal tubule (stop flow pressure, P _sf) is that filtration ceases in the blocked nephron. Should filtration not cease, but continue at a rate equal to tubular reabsorption between the block and the glomerulus, P _sf, for a given P _gc, will depend on the distance between block and glomerulus. This would have serious consequences for the interpretation of P _sf, particularly in respect of its frequent use in analysis of the tubuloglomerular feedback (TGF) mechanism. Experiments were performed in anaesthetized Wistar rats to examine whether a length dependency of P _sf exists and, if so, to what extent this relationship alters during maximal TGF stimulation by loop of Henle perfusion. A length dependency of P _sf existed both in the absence and presence of loop flow. The regression coefficients were significantly different from 0 and from each other. P _gc cannot thus be equated with the sum of P _sf and _art. The length dependent error in P _sf makes it unsuitable for the quantitative analysis of TGF and glomerular haemodynamics.     

Acute Phase Responses and Cytokine Secretion in Chronic Fatigue Syndrome

This study addresses the hypothesis that clinical manifestations of chronic fatigue syndrome (CFS) are due in part to abnormal production of or sensitivity to cytokines such as interleukin-1 (IL-1) and IL-6 under basal conditions or in response to a particular physical stress: 15 min of exercise consisting of stepping up and down on a platform adjusted to the height of the patella. The study involved 10 CFS patients and 11 age-, sex-, and activity-matched controls: of these, 6 patients and 4 controls were tested in both the follicular and the luteal phases of the menstrual cycle, and the remainder were tested in only one phase, for a total of 31 experimental sessions. Prior to exercise, plasma concentrations of the acute phase reactant ₂-macroglobulin were 29% higher in CFS patients (P < 0.008) compared to controls. Secretion of IL-6 was generally higher for CFS patients (~38%), however, this difference was statistically significant only if all values over a 3-day period were analyzed by repeated-measures ANOVA (P = 0.035). IL-6 secretion correlated with plasma ₂-macroglobulin in control subjects at rest (R = 0.767, P = 0.001). Immediately after exercise, the CFS patients reported greater ratings of perceived exertion (P=0.027) compared to the healthy control subjects. Ratings of perceived exertion correlated with IL-1 secretion by cells from healthy control subjects (R = 0.603, P = 0.022), but not from CFS patients, and IL-1 secretion was not different between groups. Exercise induced a slight (<12%) but significant (P = 0.006) increase in IL-6 secretion, but the responses of the CFS patients were not different than controls. Furthermore, no significant exercise-induced changes in body temperature or plasma ₂-macroglobulin were observed. These data indicate that under basal conditions, CFS is associated with increased IL-6 secretion which is manifested by chronically elevated plasma ₂-macroglobulin concentrations. These modest differences suggest that cytokine dysregulation is not a singular or dominant factor in the pathogenesis of CFS.