Neuronal mechanisms for pitch analysis in the time domain

Summary Many units in the auditory midbrain nucleus (MLD) of the Guinea fowl are found to be tuned to amplitude modulated tones (AM). For a given response maximum the relationship of the period _m of the modulation frequency f_m and the period _c of the carrier frequency f_c may be given by an empirical equation: m · _m + n · _c = 1 · ₁, where m, n and 1 are small integers typical for a unit. ₁ is a time constant of 0.4 ms. The temporal pattern of the neuronal response support these findings. The averages of spike trains oscillate with periods multiple to ₁. These oscillations are elicited by stimulus onsets and zero crossings of f_m and may be coupled strongly to f_m depending on f_c. Variation of f_m or f_c shifts the mean delay of the phase coupled activity proportional to m · _m and n · _c, respectively. These effects may be explained with activity phase coupled to f_c which coincides at the level of the recorded units with oscillations coupled to f_m. This is expressed by the above given periodicity equation. Psychophysical results with AM-stimuli indicate that the mechanisms described and the periodicity equation are adequate for the explanation of the analysis of periodicity pitch in humans. Hence the period corresponding to pitch is defined by m · _m + n · _c = 1 · ₁, where n and 1 are integers and ₁ = 0.4 ms. Plots of _p as a function of _c reveal steps at 0.4 ms intervals indicating that the neuronal time constant is the same in both species.Supported by the DFG, SFB 45     

Gating kinetics of ATP-sensitive single potassium channels in myocardial cells depends on electromotive force

ATP-sensitive single-channel potassium currents were studied in the membrane of rat ventricular myocytes. With an internal K⁺ concentration of [K⁺]_i=140 mM, the outwardly directed currents saturated at 1.8 pA in the region of positive potentials independently of the external K⁺ concentration [K⁺]_o, whereas an increase in [K⁺]_i of up to 300 mM caused a positive shift in the region of current saturation (from +40 mV to +100 mV) and an increase in the level of the saturation up to 4 pA. The openings of the channels appeared in bursts. Gating kinetics within the bursts were investigated. It was shown that the channel mean open (_o) and closed (_c) times during a burst depended primarily on the electromotive force (V-V _k) for potassium ions. For different [K⁺]_o, _o was maximal and _c was minimal in the region of reversal potentials (V _rev); _o decreased and _c increased gradually with deviation ofV fromV _rev. Therefore we conclude that the gating properties of the ATP-sensitive K channels depend on the ion flux parameters.     

Inhibitory synaptic channels activated by γ-aminobutyric acid (GABA) in crayfish muscle

Small crayfish muscle fibres were voltage clamped and synaptic current elicited by superfused GABA solutions was measured. Analysis of the fluctuations of synaptic current and of relaxations of the current after voltage steps yielded analogous results. The current has two components. The first component is characterized by the opening of synaptic channels with a single channel conductance =9 pS and an average open time =5 ms, measured at 23°C and-100 mV. depends on the membrane potential, _E = ₀ · e ^E/, and was about +100 mV in the average. The channel open time agrees with the time constant of decay of the inhibitory postsynaptic current (IPSC) elicited by a nerve stimulus. The current is carried by chloride ions. The second current component is much slower, the average channel open time was _s = 33 ms at 23°C and-60 mV. The open time _s of the slow component also was shortened on hyperpolarization. The reversal potential for the current component was more positive than-50 mV. This slow component also seems to be a synaptic one.This investigation was supported by a grant of the Deutsche Forschungsgemeinschaft     

Endothelial cell dynamics under pulsating flows: significance of high versus low shear stress slew rates (d(tau)/dt)

Shear stress modulates endothelial cell (EC) remodeling via realignment and elongation. We provide the first evidence that the upstroke slopes of pulsatile flow, defined as shear stress slew rates (positive / affect significantly the rates at which ECs remodel. We designed a novel flow system to isolate various shear stress slew rates by precisely controlling the frequency, amplitude, and time-averaged shear stress _ave of pulsatile flow. Bovine aortic endothelial cell (BAEC) monolayers were exposed to three conditions: (1) pulsatile flow (1 Hz) at high slew rate (293 dyn/cm² s), (2) pulsatile flow (1 Hz) at low slew rate (71 dyn/cm² s), and (3) steady laminar flow at /t=0. All of the three conditions were operated at _ave=50{dyn/cm}². BAEC elongation and alignment were measured over 17 h. We were able to demonstrate the effects of shear stress slew rates /t on EC remodeling at a fixed spatial shear stress gradient (/x). We found that pulsatile flow significantly increased the rates at which EC elongated and realigned, compared to steady flow at /t=0. Furthermore, EC remodeling was faster in response to high than to low slew rates at a given tau _ave © 2002 Biomedical Engineering Society. PAC2002: 8719Tt, 8719Rr     

Postsynaptic actions of ethanol and methanol in crayfish neuromuscular junctions

Actions of ethanol and methanol on excitatory postsynaptic channels activated by quisqualate were investigated in opener muscles from the first walking leg and the claw of crayfish. Both ethanol and methanol reduced the elementary currents |i| that flow through channels operated by quisqualate in a concentration-dependent manner but did not affect the apparent mean open time, _noise, of the channels estimated from power spectra. 0.26 mol/l ethanol, or 1 mol/l methanol, respectively, reduced |i|e-fold. Ethanol also markedly decreased the size and the decay time constant (sEPSCs) of spontaneous excitatory postsynaptic currents (sEPSCs). At ten fibres, on the average, 0.26 mol/l ethanol decreased (sEPSCs) by a factor 1.56±0.24 (SD). (sIPSCs) and _noise of inhibitory postsynaptic currents apparently were not affected by ethanol. Moreover the size of elementary inhibitory postsynaptic currents did not decrease in the presence of this alcohol. Thus, in crayfish opener muscles ethanol seems to selectively depress excitatory postsynaptic currents.This investigation was supported by the Deutsche Forschungsgemeinschaft, Project Fi 305/1-1/1-2     

Urinary excretion of histamine and some of its metabolites in man: Influence of the diet

Urinary excretions of histamine,N -methylhistamine andN -methylimidazoleacetic acid have been determined in 10 normal subjects on 3 different diets, containing a very low protein, a low protein and a high protein amount. Foodstuffs which could contain histamine were excluded. The mean excretion ofN -methylhistamine on the second day of each diet amounted to 0.861 mol/24 h, 1.051 mol/24 h and 1.378 mol/24 h, respectively. The excretions of histamine andN -methylimidazoleacetic acid were not affected.In 6 normal persons on a protein low diet, the excretions of histamine,N -methylhistamine andN -methylimidazoleacetic acid have been determined for 10 days. On the fifth day, to 3 persons 200 mol of histamine was given orally, the other 3 persons received a high protein diet. The persons receiving histamine showed a strongly enhanced excretion ofN -methylimidazoleacetic acid, corresponding to 36.1% of the administered histamine, whereas the urinary excretions of histamine andN -methylhistamine were only slightly elevated. On the high protein diet, only the excretion ofN -methylhistamine was slightly elevated.The urinary excretions of histamine in the female subjects sometimes showed unexpectedly high values. Most probably, this phenomenon is attributable to bacterial histamine production in the urogenital tract.To whom correspondence should be addressed.     

Temperature experiments on nerve and muscle membranes of frogs

The influence of temperature changes in the range of 25°C to –6°C on the time constants of Na activation (m) and inactivation (h) was studied in twitch muscle fibers and the node of Ranvier under voltage-clamp conditions. Arrhenius plots of m and h exhibit a change in activation enthalpy at temperatures below 10°C. Cooling and subsequent heating induce a hystersis in the temperature dependence of m and h Ni²⁺ and UO ₂ ²⁺ increase the hysteresis width. With fast temperature changes the gating kinetics relax to their new values more slowly than the temperature change. Hence, temperature must be changed more slowly than 5°C/min if an additional apparent hysteresis due simply to this relaxation is to be avoided. The data are explained by the hypothesis of a phase transition in the membrane lipids. This conception is favoured over a temperature-induced change in protein conformation, since the neutral local anaesthetic benzocaine shows use-dependent block as if low temperature restricted the access of the drug through the lipid phase to its receptor.Supported by grant NS 08174 from the U.S. Public Health Service and SFB 38 from Deutsche Forschungsgemeinschaft     

Quantal stores of excitatory transmitter in nerve-muscle synapses of crayfish evaluated from high-frequency asynchronous quantal release induced by veratridine or high concentrations of potassium

At single voltage-clamped opener muscle fibres of crayfish claw, 10–100 mol/l veratridine increased within a few seconds the rate of asynchronous quantal release, ñ, of excitatory transmitter from ñ<1 quantum/s to ñ10,000 quanta/s. Thereafter ñ declined exponentially either with a single, (2)50 s, or with two time constants (1)19 s, (2)50 s. In total (t), about 0.3 million quanta were released by veratridine in a single short fibre of about 1 mm length. These values were estimated by means of the noise analysis technique and they agreed with equivalent parameters of release when 100 mmol/l K⁺ were used as release stimulus. Strong quantal release could be elicited only once in a single muscle by veratridine. Furthermore, the effect of veratridine on quantal release could be completely prevented by pretreatment with tetrodotoxin. In another nerve-muscle preparation of crayfish, the abdominal superficial extensor muscle, up to 3 million excitatory quanta could be released by veratridine in a single fibre. In the latter muscle veratridine-induced asynchronous quantal release was strongly dependent on the extracellular concentration of Ca²⁺ whereas in the claw opener dependence of quantal release on extracellular Ca²⁺ was negligible.This investigation was supported by the Deutsche Forschungsgemeinschaft, SFB 220     

Measurement of urinary Nτ-methylhistamine excretion: Correlation of a newly developed radioimmunoassay (RIA) with gas chromatography mass spectrometry (GCMS)

A newly developed radioimmunoassay (RIA, Y) for the determination of urinary N-methylhistamine concentrations was correlated with gas chromatography mass spectrometry (GCMS, X). In 34 urine samples, with histamine and N-methylhistamine levels within our reference values, the correlation was: Y=1.47X–0.245 mol/l (r=0.92;p-slope 0.0001). In 14 pathological urine samples, derived from patients with mastocytosis and having upper reference values, the correlation was: Y=1.75X–1.02 mol/l (r=0.93;p-slope 0.001). In spite of the greater specificity of the monoclonal antibody for N-methylhistamine compared with that of histamine, relatively high urinary histamine concentrations gave a false positive influence on the RIA results, which was 100% when the histamine/N-methylhistamine ratio was about 19. Clear cases of mastocytosis can be diagnosed, using the RIA-kit, but for a more precise N-methylhistamine value GCMS analyses will remain necessary.     

On the temperature dependence of the Na pump in sheep Purkinje fibres

The temperature dependence of cardiac active Na transport is studied in voltage clamped sheep Purkinje fibres by means of simultaneous measurements of the membrane current (I) and the intracellular Na activity (a _Na ⁱ ). During activation of the Na pump a transient outward current (I) anda _Na ⁱ decline exponentially with an identical time constant (). The transient outward current and the decline ina _Na ⁱ are blocked by 10^–4 M dihydroouabain (DHO). Lowering the temperature from 42°C to 17°C prolongs . The electrogenic fraction (e.f.) of the active Na efflux remains unaffected. The Q₁₀ value of the active Na transport derived from the changes of varies within the temperature range studied. The Q₁₀ amounts to 1.2 between 42°C and 35°C, to 2.4 between 35°C and 22°C and to 2.1 between 35°C and 17°C. Correspondingly the activation energy of the active Na transport is not constant between 42°C and 17°C. It is calculated to be 3.4 kcal/mol between 42°C and 35°C, 15.9 kcal/mol between 35°C and 22°C and 12.4 kcal/mol between 35°C and 17°C. Variations in temperature change the maximal rate constant of the active Na transport, whereas the sensitivity of the Na pump towards the extracellular K concentration (K_o) is little affected. The unidirectional active Na efflux of a fibre as a function of the intracellular Na concentration (Na_i) at 35°C and 22°C is derived from the experemental data. The relationship is linear over the narrow Na_i range studied but seems to be more complex when a wider Na_i range is considered.Supported by the Deutsche Forschungsgemeinschaft (SFB 114 Bionach)     

End-plate currents evoked by paired stimuli in frog muscle fibres

Using voltage-clamp techniques spontaneously occuring miniature end-plate currents (mepc) and nerve-evoked end-plate currents (epc) were recorded in frog glycerol-treated or cut muscle preparations. Epcs were induced by pairs of stimuli (the delay of the 2nd stimulus, t being 6 ms–30 s; one pair was delivered every 60–90 s). The decay time constant of the epc (_epc) was longer, the larger its quantal content despite the presence of active acetylcholinesterase (AChE). After treatment with anticholinesterases (prostigmine or armin, an irreversible inhibitor) this increase in _epc became more pronounced. When AChE was fully active the decay of the 1st epc ₁ was slightly faster than the decay of the 2nd epc ₂ only when the interstimulus interval was rather short (t<20 ms). Following treatment with anticholinesterases this difference between ₂ and ₁ could be determined even when t was as long as 30 s. In anticholinesterase-treated preparations was found to be inversely proportional to log t: a 50% increase in the decay time-constant of the 2nd epc occurred with t=120 ms. During continuous stimulation (10 impulses/s) _epc increased from the 1st to the 5–6th responses, but then decreased in parallel with the fall in the epc amplidude. The phenomenon of postsynaptic potentiation we observed could be readily abolished when quantal content was decreased by the presence of magnesium ions, but it was relatively unaffected when the receptor density was decreased by -bungarotoxin (-BuTX).The possible existence is discussed of two kinds of repetitive binding of ACh molecules, first, to free cholinoreceptors (a process which could be inhibited by -BuTX) and, second, to a complex of the cholinoreceptor plus one molecule of ACh (a process which is less sensitive to -BuTX blocking action).     

Effects of hypoxia on passive electrical properties of canine ventricular muscle

The effect of hypoxia, either in the presence or in the absence of glucose, on the passive electrical properties of canine ventricular muscle fibers was examined, employing a single sucrose gap method. The significant changes after 30 min of hypoxia (PO ₂=35–45mm Hg) were an increase in the specific internal longitudinal resistance (R _i) and a decrease in the space constant (). The values during the control (PO ₂>450mm Hg) were 198±77 cm forR _i and 0.81±0.15 mm for , and they changed to 245±90 cm and 0.70±0.10mm, respectively, after 30min of hypoxia. Hypoxia decreased the specific membrane resistance (R _m), but the changes were not statistically significant. The membrane time constant ( _m ) and capacity (C{imm}) were not affected significantly. The absence of glucose during hypoxia was found to cause more profound changes than hypoxia alone in the passive electrical properties, especiallyR _i and , suggesting that glucose might counteract the effects of hypoxia on these parameters of ventricular muscles.     

Two types of transient outward currents in cardiac ventricular cells of mice

Ventricular cells of adult mice were prepared by an enzyme digestion procedure. Single channel currents were recorded by a conventional patch clamp technique from cell attached patches. Voltage steps from the holding potential of –80 mV to test potentials between –35 and +50 mV caused openings of two types of outward currents through single channels with the conductances of 27 and 12 pS, respectively. The averaged currents reveal transient time courses for both channel types. The current-voltage relations of both single channel currents were linear over the tested voltage range and intersected the voltage axis at –70 mV. This indicates that both single channel currents are mainly carried by potassium ions. All open and closed times were found to be voltage independent. The 27 pS channel had a mean open time of 3.9±1.0 ms (n=8). The closed time consisted of two components with ₁ = 2.1 ± 0.2 ms and ₂ = 50 ± 19 ms (n=8). The 12 pS channel had a mean open time of 34.0±5.2 ms (n=3) and the two components of the mean closed time have been calculated as ₁ = 8.3 ± 2.1 ms and ₂ = 120 ± 50 ms (n=3; all mean ±SD).     

Measurement ofN τ-methylhistamine concentrations in plasma and urine as a parameter for histamine release during anaphylactoid reactions

N -methylhistamine concentrations in plasma and urine were determined using a newly developed simultaneous determination for histamine andN -methylhistamine, based on isotope dilution mass fragmentography. Three groups of patients were investigated: patients receiving intravenously-administered iodamide for excretory urography, patients receiving a wasp-sting challenge, and patients treated with an intravenously-administered muscle relaxant. In all patients showing a distinct systemic anaphylactic or anaphylactoid reaction histamine andN -methylhistamine concentrations were found to be elevated. From the results of this study it can be concluded thatN -methylhistamine in plasma and urine is a good parameter for histamine release, and that the determination of this histamine metabolite are less hampered by possible artefacts (due to basophil disrupture, a very short half-life time or bacterial production) than determinations of histamine itself.     

Characterisation, asymmetry and reproducibility of on- and off-transient pulmonary oxygen uptake kinetics in endurance-trained runners

The purpose of this study was three-fold: (1) to characterise both the on- and off-transient oxygen uptake (O₂) kinetics in endurance runners during moderate-intensity treadmill running; (2) to determine the degree of symmetry between on- and off-transients; and (3) to determine the reproducibility of O₂ kinetic parameters in endurance runners. Twelve endurance-trained runners [mean (SD) age 25.2 (4.7) years, body mass 70.1 (9.7) kg, height 179.5 (7.5) cm, ventilatory threshold (V_T), 3,429 (389) ml.min^–1, maximal O₂ (O_2max) 4,138 (625) ml.min^–1] performed two multiple square-wave transition protocols on separate days. The protocol consisted of six (three transitions, 15 min rest, three transitions) square-wave transitions from walking at 4 km.h^–1 to running at a speed equivalent to 80% of the O₂ at the V_T (80%V_T). To determine the reproducibility, the protocol was repeated on a separate day (i.e. a test-retest design). Pulmonary gas-exchange was measured breath-by-breath. The O₂ data were modelled [from 20 s post-onset (or offset) of exercise] using non-linear least squares regression by a mono-exponential model, incorporating a time delay. The on- and off-transient time constants (_on and _off), mean response times (MRT_on and MRT_off) and amplitudes (A_on and A_off) were obtained from the model fit. On- and off transient kinetics were compared using paired t-tests. The reproducibility of each kinetic parameter was explored using statistical (paired t-tests) and non-statistical techniques [95% limits of agreement (LOA, including measurement error and systematic bias) and coefficient of variation (CV)]. It was found that the _on [12.4 (1.9)] was significantly (P<0.001) shorter than _off [24.5 (2.3) s]. Similarly, MRT_on [27.1 (1.9) s] was shorter than MRT_off [33.4 (2.2) s]. With respect to the reproducibility of the parameters, paired t-tests did not reveal significant differences between test 1 and test 2 for any on- or off-transient O₂ kinetic parameter (P>0.05). The LOA for _on (1.9 s), _off (2.3 s), MRT_on (1.2 s), MRT_off (3.2 s), A_on (204 ml.min^–1) and A_off (198 ml.min^–1) were narrow and acceptable. Furthermore, the measurement error (range, 4.3 to 15.1%) and CV (1.3 to 4.8%) all indicated good reproducibility. There was a tendency for _off to be more reproducible than _on. However, MRT_on was the most reproducible kinetic parameter. Overall, the results suggest that: (1) a multiple square-wave transition protocol can be used to characterise, reproducibly, both on- and off-transient O₂ kinetic parameters during treadmill running in runners; (2) the phase II time constant is independent of O_{2 max}, and (3) asymmetry exists between on- and off transient O₂ kinetic parameters.     

Einstellzeit der Pt-Elektrode bei Messungen nicht stationärer O2-Partialdrucke

Zusammenfassung Das Meßsignal bei sprunghaftenpO₂-Änderungen wird anhand des Diffusionsfeldes der Elektrode beschrieben. Es wird das zeitliche Verhalten des Meßsignals von blanken und membranbespannten Elektroden in gasförmigen und nicht gasförmigen Meßmedien betrachtet. Aus dem Verhalten des Meßsignals kann jeweils die Einstellzeit alssystematischer Meßfehler abgeleitet werden. In nicht gasförmigen Medien (z. B. biologisches Gewebe) übersteigt das Meßsignal nach einempO₂-Sprung zu höheren Werten das stationäre Endsignal. Daraus ergibt sich eine besondere Betrachtung der Einstellzeit in solchen Medien.Die Einstellzeit für Pt-Elektroden mit einfacher und doppelter Membran wird explizit angegeben. Schließlich wird für biologische Medien die Einstellzeit mit dem Diffusionsfehler [8] verglichen. Die Forderungen an eine Membran der Pt-Elektrode mit kleiner Einstellzeit und gleichzeitig kleinem Diffusionsfehler sind zusammengestellt.

---

Erklärung der Symbole a Verhältnis der Diffusionskoeffizienten zweier Membranen - Bunsenscher Löslichkeitskoeffizient des Mediums - _m Bunsenscher Löslichkeitskoeffizient der Membran - b Verhältnis der Diffusionsleitfähigkeiten von Membran und Medium - C ₁,C ₂ Proportionalitätskonstanten zwischen Meßsignal und O₂-Partialdruck - D Diffusionskoeffizient des Mediums - D _m,D _m Diffusionskoeffizienten der Membranen - Diffusionskoeffizient der effektiven Membran - DF Diffusionsfehler - DGl Differentialgleichung - d _m,d _m Dicke der Membranen - Dicke der effektiven Membran - dimensionsloser Parameter des Diffusionsfehlers - erf Fehlerfunktion - exp Exponentialfunktion - F Faradaykonstante - grad Gradient - I stationäres Meßsignal vor dempO₂-Sprung - I stationäres Meßsignal nach dempO₂-Sprung - I(t), I(),I() instationäres Meßsignal als Funktion der Zeit bzw. zeitabhängiger dimensionsloser Parameter - K Diffusionsleitfähigkeit des Mediums - K _m Diffusionsleitfähigkeit der Membran - Diffusionsleitfähigkeit der effektiven Membran - dimensionsloser Parameter - n Summationsindex - pO₂ O₂-Partialdruck - pO₂ als Funktion von Ort und Zeit bzw. zeitabhängiger dimensionsloser Parameter; Diffusionsfeld der Elektrode - p _c konstanterpO₂ vor dempO₂-Sprung - p _c konstanterpO₂ nach dempO₂-Sprung - p(r ₀+d _m , ) pO₂ an der Grenze Membran/Medium in Abhängigkeit des Zeitparameters - p(r,o) Diffusionsfeld zum Zeitpunkt (t=0) despO₂-Sprunges - p(r₀+d_m, o) pO₂ an der Grenze Membran/Medium zum Zeitpunkt despO₂-Sprunges - R Radius der ebenen, kreisförmigen Elektrode - r ₀ Radius der Elektrode mit halbkugelförmiger Pt-Oberfläche - r Kugelkoordinate - ₁,₂ dimensionslose ortsabhängige Parameter - T ₉₀,T ₉₅ Zeit, bis 90% bzw. 95% des Signalunterschiedes nach dempO₂-Sprung ausgeglichen sind (Einstellzeit) - T ₉₀,T ₉₅ Einstellzeit der Elektrode mit Doppelmembran - T _90*,T _95* Zeit, bis sich das Signal nach Übersteigen des stationären Endwertes diesem auf 10% bzw. 5% angenähert hat - dimensionslose Parameter zu den vorangegangenen Einstellzeiten - t Zeitkoordinate - , dimensionslose zeitabhängige Parameter - t _max, _max Zeit maximaler Signalhöhe nachpO₂-Sprung und zugehöriger dimensionsloser Parameter - V(t) Diffusionsgesamtfluß zur Pt-Oberfläche - Stromdichtevektor der diffundierenden O₂-Moleküle - x, y, z Kartesische Koordinaten - Integrationsvariable - ² Laplace-Operator - partielle Ableitung nach der Zeit - Integral über eine Fläche - gerichtetes Flächenelement     

Interactions of bradykinin,calcium, G-protein and protein kinase in the activation of phospholipase A2 in bovine pulmonary artery endothelial cells

Rise in free cytosolic calcium concentrations [Ca²⁺]_i in response to bradykinin and guanosine 5-O-thiotriphosphate (GTPS) was related to the action of phospholipase A₂ (arachidonic acid release). At 900 M extracellular CaCl₂, bradykinin induced a typical Ca²⁺ movement consisting of an initial [Ca²⁺]_i peak at approximately 400 nM followed by a sustained increase in the steady-state cytosolic Ca²⁺ level at approximately 290 nM. As the extracellular CaCl₂ concentration was reduced to 100 M, the bradykinin induced initial spike was reduced followed by only a marginal increase in steady-state cytosolic Ca²⁺ levels. Treatment of endothelial cells with saponin (0.002% w/w) did not increase [Ca²⁺]_i and saponin treated cells exhibited a very similar pattern of Ca²⁺ mobilization in response to bradykinin. However, with saponin treatment, GTPS (100 M) increased [Ca²⁺]_i at an almost identical tracing exhibited with 50 nM bradykinin stimulation (in either the presence or absence of 0.002% saponin). No additive increase in [Ca²⁺]_i was observed in cells stimulated with both 100 M GTPS and 50 nM bradykinin or in bradykinin stimulated cells subsequently exposed to GTPS. Pertussis toxin (PTX) did not affect the bradykinin induced Ca²⁺ mobilization. However, as we showed previously [1], PTX inhibited bradykinin stimulated arachidonic acid release. These results indicate transduction of the bradykinin signal by G-protein for both phospholipase A₂ (PLA₂) activation and Ca²⁺ mobilization but likely by different G subunits, a PTX sensitive and an insensitive subunit. Furthermore, the bradykinin and GTPS stimulated release of arachidonic acid appears to be only partially dependent on [Ca²⁺]_i. For example, 10 M ionomycin, a calcium ionophore, did not release arachidonic acid at extracellular CaCl₂ concentrations below 300 M while GTPS stimulated a greater release of arachidonic acid at 300 and 100 M CaCl₂ than at 900 M CaCl₂. However, at 100 M CaCl₂, ionomycin increased [Ca²⁺]_i to the same level as bradykinin or GTPS stimulated cells incubated in 900 M CaCl₂.In previously published experiments [1], we showed that phorbol 12-myristate 13-acetate (TPA) augments bradykinin activated arachidonic acid release in endothelial cells. In the absence of bradykinin, TPA had little effect on arachidonic acid release by endothelial cells. However, in the saponin treated cells, TPA alone (in the absence of bradykinin) caused a marked release of arachidonic acid. The bradykinin and TPA activated arachidonic acid releases were additive. The TPA activated release did not require an increase in [Ca²⁺]_i and occurred in the absence of any added extracellular CaCl₂. TPA did not induce an increase in [Ca²⁺]_i in either saponin treated or untreated endothelial cells. This TPA stimulated release of arachidonic acid was totally down-regulated by an 18 h preincubation of the cells in 500 nM TPA but was not inhibited by protein kinase C inhibitor H7.     

Sustained spontaneous activity of ranvier nodes induced by the combined actions of TEA and lack of calcium

Summary Superfusion of a Ranvier node ofr. esculenta andxenopus l. with Ringer's of reduced Ca⁺⁺-content (0.05 to 0.1 mM/l) containing 5 mM/l TEA, induced spontaneous activity for more than 30 min. The frequency of discharge at beginning of superfusion was 192±39/sec. It declined within 30 to 60 sec to a steady value of 76±26/sec. Such membranes were submitted to voltage clamp analysis: Depolarisations betweenV=0 and 20 mV produced initial, between 0 and 10 mV steady state inward currents. This reversal of steady state currents compared to normal membranes is due to enhanced Na⁺-permeability (lack of Ca⁺⁺) and to reduced K⁺-permeability (action of TEA). Analysis of the sodium system revealed (a) shift of them (V) curve by 10 mV to smaller depolarizations (b) shift of theh (V) curve in same direction by 5 mV (c) little change of time constants _m and _k. The productm ² h near the resting potential differs from zero. Spontaneous activity can thus be explained by the following cycle: After repolarization of the spike the nearly abolished sodium permeability grows toward its steady state value (hh ,mm ). Accordingly the sodium inward current increases with the evolution ofh and produces a depolarization (pacemaker potential) which by the early rise of the variablem becomes regenerative and leads to the next spike.This paper has been presented in a short note to the French Physiological Society in Milan, J. de Physiologie (Paris)59, 349 (1967).     

Analysis of miniature spontaneous inhibitory postsynaptic currents (sIPSCs) from current noise in crayfish opener muscle

Deviating from the normal situation, some crayfish muscle fibres showed spontaneous inhibitory activity: discharge of large inhibitory postsynaptic currents, IPSCs, alternating with long lasting bursts of current noise. Analysis of the bursts of current noise revealed that they are composed of spontaneous miniature unit currents, sIPSCs. In the burst periods the sIPSCs occurred with an average rate of 3.5–10 kHz and had an amplitude of about ã=90 pA at a driving force E=10 mV. The peak conductance _ã=ã/E of the sIPSCs was _ã=9.2 nS±0.5 (S.D.,n=5) for membrane potentials between E=–60 mV and E=–80 mV. _ã seemed to decrease when the membrane was hyperpolarized. The time constants of decay, , of the sIPSCs were identical with of the IPSCs. Further, and its potential dependence agreed with the mean lifetimes of inhibitory postsynaptic channels operated by -aminobutyric acid (GABA) [Dudel et al. 1977, 1980]. Synchronized opening of about 750 inhibitory synaptic channels generates a sIPSC.Analysis of this anomalous bursting inhibitory activity thus yields the size of the inhibitory quantum of transmission, which could not be obtained from IPSCs.This investigation was supported by the Deutsche Forschungsgemeinschaft