The volume-activated chloride current in human endothelial cells depends on intracellular ATP

We have studied the effect of intracellular ATP on volume-activated Cl^–-currents in endothelial cells from human umbilical veins by means of the whole-cell patch clamp technique. The run-down of this current in ruptured patches during repetitive applications of hypotonic solutions (HTS) could be significantly reduced if the cells were internally perfused with a pipette solution that contained 4 mmol/l ATP. This run-down was much less pronounced if currents were recorded using nystatin-perforated patches. The amplitude of the current was drastically reduced and its activation became slower if the cells were superfused with a glucose-free medium with 1 mmol/l KCN. Adding 4 mmol/l ATPS, a poorly hydrolyzable ATP-analogue, to the patch pipette prevented run-down of the current during repetitive activations by HTS, even if the cells were superfused with glucose-free solution with 1 mmol/l KCN. It is concluded that activation of the mechanosensitive Cl^– conductance in human endothelial cells requires the presence of intracellular ATP, but not its hydrolysis.     

Features of 4-aminopyridine sensitive outward current observed in single smooth muscle cells from the rabbit pulmonary artery

The 4-aminopyridine (4AP) sensitive outward current of enzymatically dispersed single smooth muscle cells of the rabbit main pulmonary artery were investigated using the voltage clamp method. When the cell was exposed to physiological salt solution (PSS) in the bath and high K⁺ in the pipette no inward current was generated by depolarization of the membrane, but when 4AP was present in the bath or when Cs⁺ with tetraethylammonium⁺ (Cs⁺-TEA⁺) in the pipette, an inward current was generated. This current was enhanced by Ba²⁺ or high Ca²⁺ and was blocked by inorganic or organic Ca²⁺ channel blockers.The outward current was partly inhibited by the Ca²⁺ channel blockers, Ca²⁺-free or Mn²⁺ containing solution. The residual outward current was blocked by external application of 10 mM 4AP, whereas it was inhibited by half with 100 mM TEA⁺. To investigate further natures of 4AP sensitive outward current, the following experiments were done in the bath solution containing 2.5 mM Mn²⁺. The reversal potential of this outward current, estimated from the tail current, remained the same in Na⁺-deficient solution, but shifted to near the K⁺-equilibrium potential in Cl^– deficient solution. Thus, the main current carrier for the outward current seems to be K⁺, but Cl^– may participate to some extent. The amplitude of the outward current decreased slowly. However, the reversal potential was not changed, suggesting the reduction in amplitude of the outward current was not due to the accumulation of K⁺ on the outer surface of the membrane. As 4AP inhibited the outward current to a greater extent at lower than higher membrane potential levels, 4AP bound to the channel may be dislodged at higher levels. When pH of the bath solution was modified from 7.3 to 8.0, inhibitory actions of 4AP were enhanced (pKa value of 4AP=9.17). Thus, a non-ionized form of 4AP may act as a channel blocker. We conclude that in smooth muscle cells of the pulmonary artery, lack of an action potential in physiological solution may partly be due to a small inward current as well as a large contribution of the 4AP sensitive outward current.     

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     

The late component of L-type calcium current during guinea-pig cardiac action potentials and its contribution to contraction

 L-Type Ca²⁺ current (I _Ca,L) elicited during the action potential (AP) of guinea-pig ventricular myocytes exhibits an early and a late component. The whole-cell patch-clamp technique was used to characterize the process regulating the late I _Ca,L component and to assess its contribution to excitation-contraction coupling. A stepwise decrease in repolarization rate of AP-like voltage-clamp pulses led to an exponential increase in Ca²⁺ charge carried by I _Ca,L. This saturation behaviour was significantly reduced or absent when Ba²⁺ or monovalent cations were used as charge carriers, which suggests that the late component of I _Ca,L is controlled mainly by Ca²⁺-dependent processes. Simultaneously recording I _Ca,L and zero-load shortening or the internal Ca²⁺ concentration (fura-2) revealed that Ca²⁺ carried by the late component of I _Ca,L markedly contributes to the Ca²⁺ content of the sarcoplasmic reticulum (SR). Reducing the charge transfer by late I _Ca,L during a series of AP-like conditioning clamp pulses by 48% reduced the shortening amplitude during a subsequent test stimulation by 56%. This relationship was absent during long rectangular depolarizing conditioning clamps, during which Na⁺/Ca²⁺ exchange increased its influence on SR Ca²⁺ loading. The late component of I _Ca,L developed only a minor direct influence on the simultaneous cell shortening. Thus, the main contribution of the late I _Ca,L component is to supply Ca²⁺ for SR loading. Received: 5 November 1997 / Received after revision: 12 June 1998 / Accepted: 15 June 1998     

Characterization of insulin resistance in type I diabetes

Summary Insulin sensitivity was assessed using the euglycaemic clamp technique in eight type I diabetic patients (after overnight blood glucose normalization with an artificial pancreas) and in six healthy subjects. Basal insulin concentrations were higher in diabetic patients (25±4 µU/ml) than in control subjects (17±1 µU/ml;P<0.05). Insulin infusion of 0.5, 1.0, 2.0 and 5.0 mU/kg per min during subsequent 2-h periods resulted in similar mean steady-state insulin concentrations in both groups. The mean dextrose requirements during the last 40 min of each period were nevertheless decreased in diabetic patients (1.6±0.5, 3.5±0.8, 6.5±0.7, 10.2±0.7 mg/kg per min) as compared with control subjects (4.7±0.3, 8.2±0.9, 10.2±0.9, 12.4±0.9 mg/kg per min). At low insulin concentrations dextrose requirements were diminished in all diabetic subjects. At the highest insulin levels, individual dose-response curves from only four patients were within the normal range. Under basal conditions, the monocyte receptor number was significantly reduced in diabetic patients (17,500±2,800 sites/cell) as compared with control subjects (26,700±2,500 sites/cell;P<0.05), whereas there were no differences regarding empty site affinities. Receptor data did not differ in patients with normal and decreased maximal dextrose requirements.Insulin resistance is apparently a common feature of type I diabetes at serum insulin concentrations of approximately 100 µU/ml. Normalization of the insulin effect by higher insulin concentrations is not possible in all patients. Insulin antibodies at concentrations observed in this study (<0.16 mU/ml) do not contribute significantly to insulin resistance; receptor and postreceptor defects are possibly more important.Abbreviations GCIIS Glucose-controlled insulin infusion system - SSGIR Steady-state glucose infusion rate I.N. and P.A. were fellows of the Deutscher Akademischer Austauschdienst 1981/82 and 1983/84     

Proton modulation of M-like potassium current (IKx) in rod photoreceptors

The effect of extracellular pH (pH_e 6.9–8.1) and intracellular pH (pH_i 6.4–8.1) on the non-inactivating voltage-sensitive M-like potassium current (I_Kx) was studied in patch-clamped salamander rod photoreceptors. The midpoint of the I_Kx activation curve shifted by 6.6 mV per pH_e unit, with acidification producing positive shifts and alkalinization producing negative shifts. The time constant of I_Kx activation shifted with pH_e in a manner consistent with the shifts in the activation curve. Maximum conductance and gating charge were unaffected by changes in pH_e. I_Kx did not depend on pH_i. Given the importance of I_Kx in rod function, these results suggest that pH_e could affect the signal transmitted from rods by changing I_Kx activation parameters.     

A method for exceptionally low noise single channel recordings

Summary We present a method whereby, with integrating electronics, quartz patch electrodes and a novel use of silicone oil, background noise levels as low as .083 pA RMS in a 5 kHz bandwidth (4-pole Butterworth filter) have been achieved in single channel patch clamp recordings. These approaches result in much higher signal to noise ratios for single channel recording than have previously been reported and should allow many investigators to significantly reduce noise at a constant bandwidth or to increase their recording bandwidths by several kHz.     

Regulation and possible physiological role of the Ca2-dependent K+ channel of cortical collecting ducts of the rat

In the luminal membrane of rat cortical collecting duct (CCD) a big Ca²⁺-dependent and a small Ca²⁺-independent K⁺ channel have been described. Whereas the latter most likely is responsible for the K⁺ secretion in this nephron segment, the function of the large-conductance K⁺ channel is unknown. The regulation of this channel and its possible physiological role were examined with the conventional cell-free and the cell-attached nystatin patch-clamp techniques. Patch-clamp recordings were obtained from the luminal membrane of isolated perfused CCD segments and from freshly isolated CCD cells. Intracellular calcium was measured using the calcium-sensitive dye fura-2. The large-conductance K⁺ channel was strongly voltage- and calcium-dependent. At 3 mol/l cytosolic Ca²⁺ activity it was half-maximally activated. At 1 mmol/l it was neither regulated by cytosolic pH nor by ATP. At 1 mol/l Ca²⁺ activity the open probability (P _o) of this channel was pH-dependent. At pH 7.0 P _o was decreased to 4±2% (n=9) and at pH 8.5 it was increased to 425±52% (n=9) of the control. At this low Ca²⁺ activity the P _o of the channel was reduced by 1 mmol/l ATP to 8±4% (n=6). Cell swelling activated the large-conductance K⁺ channel (n=14) and hyperpolarized the membrane potential of the cells by 9±1 mV (n=23). Intracellular Ca²⁺ activity increased after hypotonic stress. This increase depended on the extracellular Ca²⁺ activity. A possible physiological function of the large-conductance K⁺ channel in rat CCD cells may be the reduction of the intracellular K⁺ concentration after cell swelling. Once this channel is activated by increases in the cytosolic Ca²⁺ activity it can be regulated by changes in cellular pH and ATP.Supported by DFG Schl 277/2-3     

Properties of voltage-gated currents of microglia developed using macrophage colony-stimulating factor

Microglia were isolated from a murine neonatal brain cell culture in which their development had been stimulated by supplementation with the macrophage/microglial growth factor macrophage colony-stimulating factor (M-CSF). Using the whole-cell configuration of the patch-clamp technique, voltage-gated membrane currents were recorded from these microglial cells. Hyperpolarization induced inward rectifying K⁺ currents, as described for microglia from untreated cultures. These currents activated negative to the K⁺ equilibrium potential and, with a strong hyperpolarization, displayed time-dependent inactivation. The inactivation was abolished when extracellular NaCl was replaced by N-methyl-d-glucamine (NMG), thereby indicating a partial block of this K⁺ conductance by Na⁺. Inward rectifying currents were also blocked by extracellularly applied Cs⁺ or Ba²⁺. They were slightly diminished following treatment with extracellular tetraethylammonium chloride (TEA) but were not affected by 4-aminopyridine (4-AP). Upon long lasting depolarizing voltage pulses to potentials positive to 0 mV, the cells exhibited a slowly activating H⁺ current which could be reduced by application of inorganic polyvalent cations (Ba²⁺, Cd²⁺, Co²⁺, La³⁺, Ni²⁺, Zn²⁺) as well as by 4-AP or TEA. Based on their kinetics and pharmacological characteristics, both currents detected on M-CSF-grown microglia are suggested to correspond to the inward rectifier and the H⁺ current of macrophages.     

Development and testing of a simple algorithm for a glucose clamp

The glucose clamp technique is widely used in clinical research studies to maintain a constant blood glucose (BG) level during metabolic perturbation. An algorithm is used to set the rate of an intravenous glucose infusion according to measured BG concentrations. Currently used clamp algorithms are unnecessarily complex. This paper describes the development and use of a new algorithm which is simpler to implement. The algorithm employs proportional and differential feedback control. Discrete BG sampling with a sampling period of 5 min is used. Computer simulation with a two-compartment model of glucose dynamics was used to refine the algorithm. A Monte Carlo approach was adopted to examine the effects of random variations in measured BG concentrations and the elapsed time between blood sampling and adjustment of the glucose infusion rate. A coefficient of variation in BG concentration of less than 4 per cent was predicted for a euglycemic clamp. This was confirmed when the algorithm was applied in clinical research studies. This degree of BG control compares well with other published algorithms.