A re-examination of the effects of lanthanum on the frog neuromuscular junction

Lanthanum (La³⁺, 0.1–2 mM) was applied to frog cutaneous pectoris muscles at 20–25°C, or at 3–5°C, and the mean amplitude and rate of occurrence, <r>, of the miniature endplate potentials (mepps) were measured as functions of time at single neuromuscular junctions. Some muscles were fixed at 3–5°C and their nerve terminals examined in the electron microscope. When 1 or 2 mM La³⁺ was applied at room temperature, <r> rose to peak values of 0.8–3.4×10³/s and then declined to <100/s after 30–60 min. When the results are corrected for the dispersion in mepp amplitudes, we estimate that 1.8×10⁶ mepps occurred in this time. If 0.1 mM La³⁺ was applied, or if 1 mM La³⁺ was removed when <r> was near its peak, then <r> remained high for at least 1 h and 4×10⁶ mepps occurred. All these mepp counts exceed the 0.7×10⁶ quanta stored in resting nerve terminals [4]. When 1 or 2 mM La³⁺ was applied at 3–5°C, <r> rose to peak values of 50–700/s and then fell to 20–200/s after 20–30 min. If the La³⁺ was then removed, <r> declined 50% over the next hour; 0.7×10⁶ mepps occurred at the junctions treated with 1 mM La³⁺, and their terminals still contained about 69% of their vesicles. Thus, vesicles can be recycled at 3–5°C. Millimolar concentrations of La³⁺ reduced the mepp amplitude by70–80% at both temperatures and abolished almost completely the depolarization produced by bath applied acetylcholine or carbachol. These effects were reversible.Supported by U.S. Public Health Service Grant NS-18354 to W. P. Hurlbut and by a grant from the Muscular Dystrophy Association of America to B. Ceccarelli     

“Run-down” of the Ca current during long whole-cell recordings in guinea pig heart cells: role of phosphorylation and intracellular calcium

We examined by a statistical approach the decrease of the Ca current (run-down) during long-lasting recordings with the whole-cell patch-clamp technique in guinea pig ventricular myocytes. The results are as follows. (1) Run-down of the Ca current (I _Ca) occurs in three phases (T1–T3). T1 (38±19 min,n=135) and T3 (35±17 min,n=23) are characterized by a slow rate of decay ofI _Ca [90±20 and 60±20 nA·cm^–2·min^–1, respectively]. T1 and T3 are separated by T2 (6±4 min,n=135) during which the current decays quickly [1200±230 nA·cm^–2·min^–1]. Between the onsets of T1 and T3,I _Ca decreases from 11±3 to 3.5±1 A/cm². (2) Normalized current-voltage relationship, reversal potential and voltage-dependencies of steady-state activation and inactivation ofI _Ca are globally shifted toward more negative potentials during the run-down process by 10–15 mV. (3)I _Ca3 measured during T3 retains the pharmacological properties (blockade by D600, NiCl₂ and CoCl₃, increase by isoprenaline and insensitivity to tetrodotoxin) of the originalI _Ca. (4) Intracellular perfusion of the nonhydrolysable ATP analogue AMP-PNP does not prevent the occurrence of T2, suggesting that a phosphorylation-dephosphorylation process is not involved in the fast run-down ofI _Ca. (5) With 0.1 mM EGTA in the pipette, addition of 3 mM ATP significantly prolongsI _Ca survival. No improvements are obtained by increasing the ATP concentration to 10 mM or replacing ATP with creatine phosphate. With 3 mM ATP present, increasing the EGTA concentration to 10–20 mM doublesI _Ca survival time. EGTA alone (10 mM) is less effective than the mixture 3 mM ATP-0.1 mM·EGTA. Intracellular perfusion with a cytoplasmic extract considerably prolongs T2 and the overallI _Ca survival. (6) The results are consistent with the hypothesis that run-down ofI _Ca can partially be explained by a rise in intracellular Ca concentration and a loss of high energy compounds. Beneficial effect of ATP might include an increased capability of the cells to either extrude or sequester intracellular Ca, and a protection against enzymatic proteolysis.Recipient of successive fellowships from the Simone et Cino del Duca and Alexander von Humboldt FoundationsThis work was supported by the Deutsche Forschungsgemeinschaft, SFB 246, Project A1     

A rapidly inactivating Ca2+-dependent K+ current in pheochromocytoma cells (PC12) of the rat

The membrane electrical properties of undifferentiated pheochromocytoma cells of the rat (PC12) were studied using both current-and voltage-clamp techniques with the use of low-resistance blunt-tipped micropipettes (patch electrodes). In the presence of tetrodotoxin (TTX, 2–3 M), a spike-like wave form with a prominent after-hyperpolarization (AHP) was recorded following brief (< 10 ms) depolarizing current pulses. The inorganic divalent cations, Cd²⁺ (0.5 mM), Mn²⁺ (4mM), and 0 mM Ca²⁺/4 mM Mg²⁺ solution prolonged the duration, attenuated the AHP, slowed the rate of repolarization, and slightly enhanced the amplitude of this wave form. A rapidly inactivating outward current was recorded in over 70% of the cells under voltage-clamp conditions. This transient current was elicited at about ±30 mV, and was blocked by tetraethylammonium (5 mM), inorganic divalent cations (Cd²⁺, 0.5 mM; Mn²⁺, 4 mM; Ba²⁺, 3 mM), and removal of Ca²⁺ (0 mM Ca²⁺/4 mM Mg²⁺) from the local perfusion medium. In addition, 4-aminopyridine (5 mM), which blocks the transient outward K⁺ current I_A in a variety of excitable cells, did not have any appreciable effect on this rapidly inactivating current. Moreover, it was possible to elicit the current at a holding potential of ±40 mV. The reversal potential of this current was ±90 mV, and shifted positively when extracellular K⁺ concentrations were elevated. It is concluded that PC12 cells have a rapidly inactivating Ca²⁺ -dependent K⁺ current. A possible explanation for the transient nature of this current may be the presence of an effective intracellular Ca²⁺ buffering (uptake or extrusion) system.     

Patch clamped responses from outer hair cells in the intact adult organ of Corti

Outer hair cells (OHCs) from the mammalian cochlea act as both sensory cells and motor cells. We report here whole-cell tight seal recordings of OHC activity in their natural embedding tissue, the intact organ of Corti, using a temporal bone preparation. The mean cell resting potential, –76±4 mV (n=19) and input conductance (10±3 nS at –70 mV) of third turn hair cells were significantly lower than have been found in isolated cells. Two main K⁺ currents in the cell were identified. One current, activated positive to –100 mV, was reduced by 5 mM BaCl₂. The other current, activated above –40 mV, was reduced by 100 M 4-aminopyridine (4-AP) and by 30 mM tetraethylammonium (TEA). Both of these currents have been also identified in recordings reported from isolated cells. On stepping to different membrane potentials, cells imaged in the organ of Corti changed length by an amount large enough to cause visible distortions in neighbouring cells. By quantifying such distortions we estimate that the forces generated by OHCs can account for the enhanced response to sound required by the cochlear amplifier.     

The cardioplegic solution HTK: effects on membrane potential,intracellular K+ and Na+ activities in sheep cardiac Purkinje fibres

The effects of the cardioplegic solution HTK on membrane potential (E_M) and intracellular K and Na activities (a _K ⁱ , a _Na ⁱ ) were studied in sheep cardiac Purkinje fibres by means of conventional and ion-selective microelectrodes. HTK contains (mM): Na 15, K 10, Ca 0, Mg 4, histidine 180, (1) In control conditions E_M was –74.3±3.3 mV (n=25), a _K ⁱ was 116.4±4.1 mM (n=7) and a _Na ⁱ was 8.2±1.4 mM (n=15). (2) Exposure to HTK led to a depolarization to –59.7±3.6 mV (n=25) which exceeded by about 5–7 mV that induced in a Tyrode solution of 10 mM K and in a modified HTK solution supplemented by 2 mM Ca (n=6). (3) Addition of 0.5 mM barium eliminated the difference in the steady-state depolarization. (4) HTK superfusion increased a _K ⁱ to 120.1±4.4 mM (n=7) and decreased a _Na ⁱ to 3.9±0.9 mM (n=15). (5) The decrease in a _Na ⁱ was insensitive to amiloride (1 mM) and to external alkalization but was slightly increased by addition of 2 mM calcium. (6) When the calcium in Tyrode solution was lowered from 2.0 mM to 0.05 mM, a _Na ⁱ hardly decreased during subsequent exposure to unmodified HTK and it increased in the presence of 0.1 mM dihydroouabain. We propose the hypothesis (1) that the difference in membrane depolarization between HTK and a 10 mM K-Tyrode is caused by a decrease in K conductance by the HTK solution and (2) that the a _Na ⁱ decline mainly results from a coupled Ca influx via Na-Ca exchange due to a delayed washout of external calcium.This work was supported by the Deutsche Forschungsgemeinschaft, SFB 330 — Organprotektion     

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.     

Effects of Ca2+ removal and of tetraethylammonium on membrane currents induced by carbachol in isolated cells from the rat parotid gland

In freshly dispersed rat parotid acinar cells, 10 M carbachol increased outward currents at 0 mV and also inward currents at –70 mV recorded with the whole-cell clamp method using patch pipettes containing 1 mM EGTA. When EGTA in the pipette was increased to 2.4 mM, carbachol increased only outward currents and a further increase of EGTA to 4 mM blocked the carbachol response. Effects of changes in external K⁺ and Cl^– concentrations suggested that outward currents were carried by K⁺ and inward by Cl^–. Effects of Ca²⁺ removal from the medium differed between experiments with 0 and 5 mM ATP in the patch pipettes. When pipettes contained no ATP, responses evoked by repeated applications of 10 M carbachol (0.5–1 min) at 1.5–4 min intervals decreased only slowly after Ca²⁺ removal, outward currents being reduced to 90±6% and inward currents to 47±11% (n=6) in 10 min. On the other hand, when 5 mM ATP was included in the electrodes, Ca²⁺ removal abolished the carbachol responses in about 5 min (n=4). It was also found that tetraethylammonium (5 mM) strongly reduced both currents, by blocking muscarinic receptors, while Ba²⁺ (2.4 mM) inhibited only the outward K⁺ current.     

An endogenous inactivating inward-rectifying potassium current in oocytes of Xenopus laevis

 An endogenous inward-rectifying K⁺ current is described, which is present in native oocytes of some Xenopus laevis donors. Experiments were performed using defolliculated oocytes from donor frogs obtained from two different suppliers. In all oocytes from animals from one source, an inward-rectifying K⁺ current could be elicited with negative pulses from a holding potential of –20 mV in external solutions with a high K⁺ concentration. Increasing external K⁺ concentrations increased the amplitude of this current and shifted the reversal potential towards more positive potentials. In 118 mM KCl, the inward-rectifying K⁺ current partially inactivated between –20 and –80 mV and completely inactivated at more negative membrane potentials; 50% steady-state inactivation occurred near –50 mV. The time course of inactivation of the inward-rectifying current could be well fitted with two exponentials. The slow time constant had values of about 500 ms and was voltage independent. In contrast, the fast time constant and the time to reach the peak inward current decreased with more negative membrane potentials. Ba²⁺, Cs⁺, quinine (all 5 mM) and 50 mM tetraethylammonium partially blocked the inward-rectifying K⁺ current, whereas 10 mM 4-aminopyridine was without blocking effect. The oxidant chloramine-T blocked the inward-rectifying K⁺ current without slowing its inactivation. Received: 21 November 1995 / Received after revison and accepted: 24 May 1996     

Quantitative localization of Na-K pump sites in the frog sacculus

Summary The Na-K pump site distribution within the macula, perimacula, and wall epithelia of the sacculus in the frog inner ear was examined with quantitative [³H]ouabain autoradiography. Excised tissue was incubated for 10–30 min (23 ° C) in micromolar concentrations of high specific activity [³H]ouabain (14–70 Ci mT^–1, 5–15 Ci mmor^–1), washed for 30 min (4 ° C), then rapidly frozen (–175 ° C) and processed for light and electron microscope autoradiography. Control experiments based on (1) high K⁺ (50 mm) in the incubation and (2) low specific activity [³H]Jouabain (1 mM, 0.013–0.025 Ci mmol^–1) indicated negligible nonspecific binding of the [³H]ouabain.Measurable levels of specific [³H]ouabain binding occurred in all saccular regions examined. Binding was localized to the basolateral cell membranes with no detectable binding to the apical membranes. [³H]ouabain binding across the apical-basal axis of the saccule macular epithelium was nonuniform. Binding was low in the apical region, rose to a peak in the middle two-thirds, and then fell again close to the basement membrane. Electron microscope autoradiography suggested that this peak was due to ouabain binding to nerve terminals. Denervation of the sacculus eliminated the peak in [³H]ouabain binding and quantitative grain density analysis revealed that 45% of the Na-K pumps within the saccule macula were located on the nerve terminals.Na-K pump site density per unit volume was estimated by quantitative grain density analysis and the following values were obtained (sites m^–3 × 10³, means ± S.E.M.): saccule macula, 1.9 ± 0.2; saccule perimacula, 1.1 ± 0.1; saccule wall, 2.3 ± 0.3. Stereological analysis of conventionally fixed tissue was used to estimate overall plasma membrane surface area per unit volume (S _v). Na-K pump site densities per unit membrane area for the various regions were calculated by combining the autoradiographical and Stereological data. The following values were obtained (sites m^–2 ± 25%): saccule macula, 2500; saccule perimacula, 2500. Values for individual cells within the macula (sites m^–2 ± 25%) were: hair cells, 3000; nerve terminals, 3000; supporting cells, 1500.     

Ion-induced release of LHRH,TRH and α-MSH from hypothalamic synaptosomes and its inhibition by vinblastine

Homogenates of rat hypothalamic tissue were fractionated by means of discontinuous sucrose density gradient centrifugation. Immunoreactive luteinizing hormone releasing hormone (LHRH), thyrotropin releasing hormone (TRH), and -melanocyte stimulating hormone (-MSH) were found to be concentrated in the synaptosome-enriched fraction. This fraction was suspended in 0.32 M sucrose and the release of the three peptides was investigated. After incubation, the synaptosomes were re-isolated by ultrafiltration, and the concentration of each peptide in the ultrafiltrate was determined by radioimmunoassay. When the synaptosomal fraction was incubated at 30° C in 0.32 M sucrose containing either 60 mM K⁺-2 mM Ca²⁺ or 140 mM Na⁺ alone a release of LHRH, TRH, and -MSH occurred. Of the total content 30–50% of LHRH but only about 10% of TRH and -MSH was releasable. When the synaptosome preparation was preincubated for 30 min at 30°C with 10^–4 M vinblastine. K⁺- as well as Na⁺-induced release of LHRH, THR, and -MSH was inhibited, and the stimulatory effect of each cation was almost totally blocked by preincubation with 5×10^–4 and 10^–3 M vinblastine. The inhibitory action of vinblastine (5×10^–4 M) did not affect the oxidation of glucose to CO₂ by the synaptosomes. The results of the present investigation demonstrate that synaptosome-enriched fractions of hypothalamic origin are more stable with respect to LHRH, TRH, and -MSH release during incubation in isotonic sucrose than they are in ionic solutions, and that the peptides are released by a vinblastine-sensitive mechanism.Supported by grants from the National Institute of Arthritis, Metabolism, and Digestive Diseases (AMO 1237), the National Institute of Child Health and Human Development (HDO8672), and the National Institutes of Health contract (5-P17-HL1487-06)Supported by Grant No. 512-6951 from the Danish Medical Research Council     

Use of the fluorescent probe 1-anilinonaphthalene-8-sulfonate to study the synaptosomal transmembrane potential

The transmembrane potential (TMP) of synaptosomes isolated from the rat cerebral cortex was studied with the aid of 1-anilinonaphthalene-8-sulfonate (ANS). Addition of valinomycin to the synaptosomes was accompanied by an increase in the intensity of fluorescence of the probe at 464 nm (_exc = 365 nm), which is interpreted as reflecting hyperpolarization of the synaptosomal membranes. A decrease in the K⁺ gradient on the synaptosomal membrane (a decrease in TMP), achieved by preincubation of the synaptosomes with ouabain (1 mM) or an increase in the concentration of extrasynaptosomal K⁺ from 5 to 20 mM appreciably reduced the effect of valinomycin. Valinomycin had no effect on synaptosomes exposed to osmotic shock. It is suggested that valinomycin-induced changes in the intensity of fluorescence of ANS be used as a test of preservation of the K⁺ gradient (the presence of a TMP) by synaptosomal preparations, i.e., of the active state of the synaptosomes.Laboratory of General Pathology of the Nervous System, Institute of General Pathology and Pathological Physiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR S. E. Severin.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 89, No. 3, pp. 300–302, March, 1980.     

Comparison of the effects of potassium and pH on the calibre of cerebral veins and arteries

The vasomotor responses of individual pial veins and arteries on the convexity of the cerebral cortex to perivascular microinjection of mock cerebrospinal fluid (CSF) containing various concentrations of potassium (K⁺) and of various pH (achieved by altering the bicarbonate, HCO ₃ ^– concentration) have been examined in cats anaesthetised with -chloralose.Microapplication of CSF containing 0 mM HCO ₃ ^– (pH 4.80) effected significant increases in calibre of pial veins and arteries of 9.3±2.4% and 38.2±4% respectively (mean calibre change ±SE), whereas CSF containing 22 mM HCO ₃ ^– (pH 7.45) which constricted pial arteries significantly (–18.5±2.9%) minimally altered venous calibre (–4.3 ±2.2%).Microapplication of CSF containing 0 mM potassium resulted in a significant reduction in pial arterial calibre (–11.4±2.8%) but failed to alter pial venous calibre (–0.3 ±0.6%). Perivascular microapplication of CSF containing moderately elevated potassium concentrations (10 mM) which effected marked, significant increases in pial arterial calibre (49.3±3.9%) did not significantly alter the calibre of the pial veins (mean response –1.6±2.4%). The perivascular administration of CSF containing a high concentration of potassium (40 mM) resulted in the significant constriction of both pial veins (–13.5±0.9%) and pial arteries (–47.2±6.3%). The magnitude of the response was significantly smaller in the pial veins.The relative insensitivity to K⁺ and pH of the pial veins as compared to pial arteries suggests that alteration in the chemical composition of the perivascular fluid are of lesser importance in the control of cerebrovascular capacitance than for the regulation of cerebrovascular resistance.     

Dose-response relationship for a positive inotropic effect of insulin on isolated papillary muscle

Summary Beneficial effect of glucose and insulin on the myocardium are still a matter of discussion. The influence of insulin on isometric force of contraction of right ventricular papillary muscles of guinea pigs war studied. The papillary muscles were mounted vertically in a 95% O₂, 5% CO₂ modified Krebs-Hensuleit solution (31.5° C, 5.5 mM glucose) and stimulated 1/s. A positive inotropic effect of insulin was dedectable at a concentration of 5×10^–4 IU/ml insulin, was half maximal (52% above controle force of contraction) at 8×10^–3 IU/ml and maximal at 10^–1 IU/ml. The maximal positive inotropic effect was observed 4.7±0.6 min after addition of insulin. After the maximum there was a decrease to a steady state level of 109.8±8.5% of control (p<0.05) in 14.6±1.3 min. Higher glucose (16.5 mM) only shifted the half maximal positive inotropic effect to 5.5×10^–3 IU/ml insulin (n.s.). Inhibition of glycolysis with hypoxia or jodoacetate (5×10^–5 M) did not prevent the positive inotropic effect as known as 75% of control force was retained. When glucose transport was blocked with phlorizin (5×10^–3 M) or phloritin (5×10^–4 M) no positive inotropic action of insulin was observed. Therefore we conclude that the positive inotropic effect of insulin in isolated papillary muscles is mediated by inhanced glucose transport.     

Na+-Ca2+ exchange in the isolated cochlear outer hair cells of the guinea-pig studied by fluorescence image microscopy

The outer hair cell isolated from the guinea-pig was superfused in vitro and the cytosolic calcium concentration ([Ca²⁺]_i) and sodium concentration ([Na⁺]_i) were measured using fluorescence indicators. Under the resting condition, [Ca²⁺]_i and [Na⁺]_i were 91±9 nM (n = 51) and 110±5 mM (n = 12), respectively. Removal of external Na⁺ by replacing with N-methyl-D-glucamine (NMDG⁺) increased [Ca²⁺]_i by 270±79% (n = 27) and decreased [Na⁺]_i by 23±4 mM (n = 6). Both changes in [Ca²⁺]_i and [Na⁺]_i were totally reversible on returning external Na⁺ to the initial value and were inhibited by addition of 0.1 mM La³⁺ or 100 M amiloride 5-(N,N-dimethyl) hydrochloride. Elevation of external Ca²⁺ ions to 20 mM reversibly decreased [Na⁺]_i by 8±6 mM (n = 5). Moreover, the chelation of the intracellular Ca²⁺ with 1,2-bis (2-aminophenoxy) ethane-N,N,N,N-tetraacetic acid (BAPTA) exerted an inhibitory action on the NMDG⁺-induced reduction in [Na⁺]_i. Exposure to 5 mM NaCN for 2 min significantly and reversibly increased [Ca²⁺]_i by 290±37% (n = 5), but did not affect the [Ca²⁺]_i elevation induced by the NMDG⁺ solution. The rise in [Ca²⁺]_i induced by the NMDG⁺ solution was not enhanced by ouabain pretreatment. Addition of ouabain did not alter the [Na⁺]_i. The present results are best explained by the presence of an Na⁺-Ca²⁺ exchanger in cell membrane and indicate that the activity of Na⁺/K⁺ pump is poor in outer hair cells.     

Hypertonic saline infusion induces activation of the lymphocyte Na+/H+ antiport and cytosolic alkalinization in healthy human subjects

The Na⁺/H⁺ antiport is a membrane transport protein that extrudes intracellular protons in exchange for extracellular sodium. Some details of its physiological and pathophysiological role remain poorly defined. Experimental evidence suggests that the antiporter is involved in the regulation of cell volume. In the present study, we therefore investigated the activity of the lymphocyte Na⁺/H⁺ antiport in nine healthy volunteers following acute hypertonic (2.5%) saline infusion (4 mmol NaCl/kg over 120 min). Antiport activity was measured after acidifying the cells with Na⁺ propionate (5–40 mM) using the fluorescent dye bis-carboxyethyl carboxyfluorescein. Hypertonic saline induced significant increases in plasma osmolality (308.4±2.3 vs. 293.5±2.7 mOsm/kg; P<0.01), serum Na⁺ (150.8±3.7 vs. 138.9±0.5 mmol/kg; P<0.01), and Cl^– concentrations (118.0±3.9 vs. 101.1±1.0 mmol/kg; P<0.01). Extracellular hypertonicity was followed by a stimulated activity of the lymphocyte Na⁺/H⁺ antiport with an increase in the apparent V _max values from 2.44^±0.16 to 3.27^±0.34 10^–3 s^–1 (P<0.01) and a slight rise in pK from 6.81±0.03 to 6.87±0.03 (P<0.05) after hypertonic saline. In addition to antiport activation, cytosolic alkalinization was observed with cytosolic pH values averaging 6.90±0.02 before and 6.99±0.02 (P<0.01) after hypertonic saline. Our results show for the first time that acute extracellular hypertonicity in man due to hypertonic NaCl loading is associated with a stimulated lymphocyte Na⁺/H⁺ antiport activity and cytosolic alkalinization.Abbreviations BCECF-AM bis-carboxyethyl carboxyfluorescein, acetoxymethyl ester - HEPES 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid     

Micro Flow-through PCR in a PMMA Chip Fabricated by KrF Excimer Laser

As the third PCR technology, micro flow-through PCR chip can amplify DNA specifically in an exponential fashion in vitro. Nowadays many academies in the world have successfully amplified DNA using their own-made flow-through PCR chip. In this paper, the ablation principle of PMMA at 248 nm excimer laser was studied, then a PMMA based flow-through PCR chip with 20 cycles was fabricated by excimer laser at 19 kv and 18 mm/min. The chip was bonded together with another cover chip at 105^∘C, 160N and 20 minutes. In the end, it was integrated with electrical thermal thin films and Pt100 temperature sensors. The temperature controllers was built standard PID digital temperature controller, the temperature control precision was ± 0.2^∘C. The temperature grads between the three temperature zones were 16.5 and 22.2^∘C respectively, the gaps between the temperature zones could realize heat insulation.National Natural Science Foundation of China (Grant No.50175002), and Beijing (Grant No. 3031001).     

Granulosa cells have calcium-dependent action potentials and a calcium-dependent chloride conductance

We have found chicken granulosa cells to be excitable. Experiments using the whole-cell patch-clamp technique showed that they had membrane resting potentials of –62±3 mV (n=8) and generated action potentials, either in response to 10-ms depolarizing current pulses or, on occasion, spontaneously. The action potentials persisted in a Na⁺-free bath and were reversibly blocked by 4 mM Co²⁺. They lasted 0.9–3.0 s with 64 mM Cl^– in the pipette, were shortened 67±8 % by the Cl^– channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB; 20 M), and lengthened to 8.7±2.2 when the Cl^– equilibrium potential (V _Cl) was changed from –20 mV to –2 mV by using 134 mM Cl^–in the pipette. With conventional whole-cell voltageclamp, slowly activating and inactivating currents, which reached maximum amplitude after 0.35–1.40 s, were evoked by depolarizing voltage steps. These slow currents activated between voltage steps of –60 mV and –50 mV and reached a maximum inward amplitude at about –40 mV. Changing the Cl^– concentration in the pipette (V _Cl of –2 mV or –20 mV) or bath (V _Cl of –2 mV or + 18 mV) shifted their reversal potential in a direction consistent with a Cl^– electrode. They were inhibited by the Cl^– channel antagonists 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS; 0.5 mM), NPPB (20 M), and 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (SITS; 0.5 mM). The slow currents were blocked by Ca²⁺ deprivation, or by Co²⁺ (4 mM), or by replacing external Ca²⁺ with Ba²⁺. They showed pronounced inward rectification when a weakly buffered 0.2 M Ca²⁺ pipette solution was used, but this rectification was much reduced when pipette solutions contained 5 M Ca²⁺. The function of this Ca²⁺-dependent Cl^– current and the physiological trigger(s) of the action potentials and their role(s) in granulosa cell function remain to be determined.     

Mechanical characteristics of chemically skinned guinea-pig taenia coli

Strips of intact and chemically skinned (Triton X-100) taenia coli were mounted for isometric and quick-release experiments at 23°C. Active force increased in repeated high-K⁺ induced contractures in the intact muscle. Stable maximal force was 313±24 mN/mm² (n=6). The skinned preparations activated by Ca²⁺, at 2 mM Mg²⁺, 3.2 mM MgATP and ionic strength 0.085 M, gave half maximal force atpCa=5.62±0.4 and a maximal force (63±8 mN/mm²) atpCa=4.5 (20–25 of the control K⁺-responses prior to skinning but about 60% of the first K⁺-response). Force-velocity relations were obtained from intact muscles and from the same muscles chemically skinned and activated at optimal Ca²⁺. Maximal shortening velocity (V _max) was unaltered in the skinned preparation compared to the intact muscle (0.138±0.011 vs 0.140±0.006 L/s) indicating similar kinetics of actomyosin interaction. In the intact muscle a decrease inV _max was found when the Ca²⁺ concentration was reduced. Calmodulin (1M) increased Ca²⁺ sensitivity (by about 0.6 log units) of the skinned preparation but at optimal Ca²⁺ caused no alteration in isometric force orV _max Apreliminary report of some of the results presented here was given at the Scandinavian Physiology Society Meeting in Århus, November 1981. Arner A, Hellstrand P (1982) Acta Physiol Scand (Abstract) 114: 38 A.     

Potassium kinetics in human muscle interstitium during repeated intense exercise in relation to fatigue

Accumulation of K⁺ in skeletal muscle interstitium during intense exercise has been suggested to cause fatigue in humans. The present study examined interstitial K⁺ kinetics and fatigue during repeated, intense, exhaustive exercise in human skeletal muscle. Ten subjects performed three repeated, intense (61.6±4.1 W; mean±SEM), one-legged knee extension exercise bouts (EX1, EX2 and EX3) to exhaustion separated by 10-min recovery periods. Interstitial [K⁺] ([K⁺]_interst) in the vastus lateralis muscle were determined using microdialysis. Time-to-fatigue decreased progressively (P<0.05) during the protocol (5.1±0.4, 4.2±0.3 and 3.2±0.2 min for EX1, EX2 and EX3 respectively). Prior to these bouts, [K⁺]_interst was 4.1±0.2, 4.8±0.2 and 5.2±0.2 mM, respectively. During the initial 1.5 min of exercise the accumulation rate of interstitial K⁺ was 85% greater (P<0.05) in EX1 than in EX3. At exhaustion [K⁺]_interst was 11.4±0.8 mM in EX1, which was not different from that in EX2 (10.4±0.8 mM), but higher (P<0.05) than in EX3 (9.1±0.3 mM). The study demonstrated that the rate of accumulation of K⁺ in the muscle interstitium declines during intense repetitive exercise. Furthermore, whilst [K⁺]_interst at exhaustion reached levels high enough to impair performance, the concentration decreased with repeated exercise, suggesting that accumulation of interstitial K⁺ per se does not cause fatigue when intense exercise is repeated.     

The effect of increasing extracellular potassium concentration on the resting heat rate of the isolated rat papillary muscle

The effect of elevating extracellular K⁺ concentration on the basal metabolism of the isolated rat left ventricular papillary muscle has been investigated. The preparation was mounted on a thermopile and connected to a force transducer, to allow simultaneous measurement of muscle heat production and force. The resting heat rate (RHR) of the quiescent preparation was measured as an index of basal metabolism. Throughout all of the experiments, the muscles were maintained under a resting force of 10 mN and all measurements of RHR were made at times when there was no active force present above this passive level. Elevating the extracellular K⁺ concentration from 5.9 to 20, 40, then 80 mM produced graded increases in the RHR. The increase in RHR produced by 40 mM K⁺ was observed to be time-dependent, its effect being significantly greater at 5–7 h than at 2–4 h after cardiectomy. Averaged over all times, the percentage increases in RHR produced by 20, 40, and 80 mM K⁺ in the presence of 2 mM Ca²⁺ were 6.4±2.0%, 28.7±2.3%, and 51.3±8.9% (mean±SEM) respectively. The high K⁺-induced increase in basal metabolism was also shown to be Ca²⁺-dependent, the increase in RHR produced by 40 mM K⁺ being greater the higher the extracellular Ca²⁺ concentration (0.5–8.0 mM). The addition of verapamil was found to partially inhibit the K⁺-induced increase in resting metabolism. These results show that elevation of the extracellular K⁺ concentration produces a graded increase in the RHR that is Ca²⁺-dependent. It is suggested that the increases in RHR observed in this investigation are in part associated with elevated levels of myoplasmic free Ca²⁺ which stimulate the internal Ca²⁺ pumps.