Curare as a neuromuscular blocking agent in insects.

Topical application of curare produced no effects on electrical activity in single flight-muscle fibers of the fly Sarcophaga. However, the intraabdominal injection of curare induced a neuromuscular block similar to that described for vertebrates. The general refractoriness of some insects to chemical agents may well be due to the method of application.

Action potentials in macrophages derived from human monocytes.

The electrical activity of macrophages derived from human blood monocytes was recorded in vitro with intracellular microelectrodes and was analyzed with computer-assisted data acquisition and analysis techniques. In cells impaled 6 to 8 days after the cultures were prepared, the resting potentials reached a maximum value of -72 millivolts. The cells were electrically excitable; spikes exhibited a slow upstroke, a fast downstroke, a discrete threshold, a large overshoot, and a brief undershoot. Repetitive firing was induced by a maintained depolarizing current. A positive relation was observed between transmembrane currents and resting potential. Voltage-current relations were nonrectifying for subthreshold current injections. Since these cells had not been treated with any specific activation factors, the electrical activity recorded is evidence for the presence of voltage-dependent inward and outward currents in the membranes of mature macrophages. The electrical signals generated by these cells may be useful for the assay of sensor and effector functions of macrophages, such as chemotaxis, receptor-ligand interactions, and phagocytosis.

Calcium spike electrogenesis and other electrical activity in continuously cultured small cell carcinoma of the lung.

Spike electrogenesis, local depolarizing and hyperpolarizing responses, spontaneous rhythmic firing, and alternating resting potentials were measured in cells from a continuously cultured small cell carcinoma of the lung. Spike generation was blocked by MnCl2. In view of this evidence for calcium-spike electrogenesis and previous evidence of secretory activity in these cells, this cell line (DMS 53) can provide a model for the study of excitation-secretion behavior in human neoplastic cells.

Mercury suppression of a potassium current in human B lymphocytes.

Mercury is a recognized environmental toxin. Several organ systems are targeted by this substance and impairment of immune function is known to result from exposure to mercury. Using the patch clamp technique in the whole cell configuration on resting human B lymphocytes we have identified an outward potassium current and studied the effects of mercury on this current. We present data that demonstrate: (i) the absence of inward currents; (ii) a time and voltage dependent outward current with a threshold of -40 mV and reversal potential near EK+; (iii) blocking of this current by TEA (tetraethylammonium chloride) in a dose dependent manner; (iv) a slow time course for recovery from inactivation of this outwardly rectifying K+ current and, (v) the diminution and final block of this potassium current by mercury. These data supplement the findings from our laboratories that demonstrate inhibitory effects on B cell activation by mercury. We propose that the movement of potassium ions across the B cell membrane, an event presumed to be one of the first signals in the mitogenic process, is a target of mercury toxicity.

Interleukin-4 activates ion channels in B lymphocytes.

The lymphokine interleukin-4 (IL-4) has been shown to induce dramatic changes in the physiology of resting B cells. We have applied the patch clamp technique in the cell attached and inside/out configurations to resting and IL-4-treated B cells to determine whether specific ion conductances result as a consequence of IL-4 action. We report here that two distinct ion channel events occur in B lymphocytes after treatment with IL-4, (i) induction of an inward rectifying K+ channel that is not observed in untreated cells, and (ii) activation of a large conductance anion channel that is normally silent in non-treated cells in the cell attached patch configuration. These data present the first evidence of a direct effect by IL-4 on ion channels and we suggest roles for these two ionic conductances in IL-4-induced B cell activation.

Effects of cadmium on potassium currents in activated B lymphocytes.

We have applied the patch clamp technique in the whole-cell configuration to study whole-cell currents in B lymphocytes under three conditions: (i) resting; (ii) interleukin-4 (IL-4)-treated; and (iii) IL-4 plus cadmium-treated murine B lymphocytes. Through these experiments we have: (i) confirmed our earlier findings and the observation of others that resting B cells express only outward currents; (ii) confirmed the presence of an inwardly rectifying K+ current elicited by treatment with the lymphokine IL-4 that was revealed in our previous study on single channel currents; (iii) demonstrated that both inward and outward rectifying K+ currents in IL-4-treated B cells are dramatically reduced by exposure to 20 microM cadmium; and (iv) determined that the activation curve of the IL-4-induced inward rectifier is shifted to more negative voltages by cadmium. We propose that one of the mechanisms by which cadmium can mediate toxicity in activated B lymphocytes is through the suppression and modulation of potassium currents, effects that may alter the timing of entry into the cell cycle.

Characterization of a large conductance non-selective anion channel in B lymphocytes.

Studies on ion channel currents in freshly isolated murine B lymphocytes with the patch clamp technique revealed the presence of a non-selective anion channel of large conductance in inside-out (i/o) patches. This channel is characterized here according to its unitary conductance, ion selectivity, regulatory factors, distribution and kinetic behaviour. With a unitary conductance of 348 +/- 4.4 pS in a normal physiological ion gradient, it exhibited an indiscriminate selectivity to cations (Na+ and K+). Selectivity to chloride over sodium was established by substitution of high concentrations of NaCl (450 mM) in the bath (i/o patches), resulting in a selectivity ratio (PCl/PNa) of 33. Selectivity to chloride over potassium was confirmed in a similar manner by substitution of TEA-Cl for KCl, yielding a selectivity ratio (PCl/Pk) greater than 80. Conductance of aspartate through the channel demonstrated the non-selective nature of this anion channel. Voltage proved to be a regulatory factor but other influences on channel activity were also present, including the configuration of the patch (channel is inactive in cell attached patches), and the enhancement of activity at negative membrane voltages by previous pulsing. Intracellular levels of calcium (i/o patches) did not appear to control channel conductances or regulate kinetic activity. Kinetic behaviour of this channel was complex, with periods of bursting and flickering activity interspersed with prolonged closed/open intervals. Multiple subconductance states were also present. The complex properties and behaviour of this channel suggest a possible role in signal transduction in B cell activation.

Ionic signals in T47D human breast cancer cells.

Increasing evidence that ion channels play a key role in the modulation of cellular mitogenesis led us to investigate the membranes of T47D human breast cancer cells to identify the ion currents present. We report here the results of voltage-clamp studies in the whole-cell configuration on isolated, non-synchronized single cells obtained from a ductal breast carcinoma. In these studies we identified an outward rectifying potassium current and a chloride current. The potassium current activated at potentials more positive than -40 mV, reached an average value of 1.4 nA, and did not inactivate with time. This current was sensitive to block by extracellular tetraethylammonium chloride (TEA, IC50 = 1 micro M), was insensitive to charybdotoxin (CTX, IC50 = 7.8 micro M), and was not diminished by repetitive pulses separated by 1 s. Rapid voltage-dependent inactivation of the current was demonstrated by tail current analysis. The current appeared calcium-insensitive. Application of hyperpolarizing pulses did not elicit an inward potassium rectifier current. Treatment with tetrodotoxin did not reveal the presence of an inward sodium current. The potassium current was increased by the presence of aspartate in place of chloride and in the presence of the chloride channel blocker 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). We conclude that currents present in T47D breast cancer cells include a chloride current and a voltage-gated potassium outward rectifier. We suggest that the potassium current, either alone or in conjunction with potassium currents reported in different human breast cancer cell lines by others, may play a role in the modulation of the cell cycle.

A computer method for the acquisition and analysis of patch-clamp single-channel currents.

The method we present instructs the computer to deliver sets of programmed membrane voltages and allows the acquisition of large amounts of digitized data, compact storage, ready identification of records and rapid, interactive analysis of channel current data. A typical cycle of analysis including amplitude determination and kinetic measurements for 10 s of continuous data digitized at 5 kHz requires 5-20 min depending on the complexity of the observed channel activity. This procedure is compatible with such storage protocols as that described by Bezanilla [Biophys. J., 47 (1985) 437-441], although there are obvious benefits to using standard computer storage devices such as hard disks, floppy disks, and Bernoulli Boxes. The programs described in this report are available from the authors.

CO2 chemosensitivity in Helix aspersa: three potassium currents mediate pH-sensitive neuronal spike timing.

Elevated levels of carbon dioxide increase lung ventilation in Helix aspersa. The hypercapnic response originates from a discrete respiratory chemosensory region in the dorsal subesophageal ganglia that contains CO(2)-sensitive neurons. We tested the hypothesis that pH-dependent inhibition of potassium channels in neurons in this region mediated the chemosensory response to CO(2). Cells isolated from the dorsal subesophageal ganglia retained CO(2) chemosensitivity and exhibited membrane depolarization and/or an increase in input resistance during an acid challenge. Isolated somata expressed two voltage-dependent potassium channels, an A-type and a delayed-rectifier-type channel (I(KA) and I(KDR)). Both conductances were inhibited during hypercapnia. The pattern of voltage dependence indicated that I(KA) was affected by extracellular or intracellular pH, but the activity of I(KDR) was modulated by extracellular pH only. Application of inhibitors of either channel mimicked many of the effects of acidification in isolated cells and neurons in situ. We also detected evidence of a pH-sensitive calcium-activated potassium channel (I(KCa)) in neurons in situ. The results of these studies support the hypothesis that I(KA) initiates the chemosensory response, and I(KDR) and I(KCa) prolong the period of activation of CO(2)-sensitive neurons. Thus multiple potassium channels are inhibited by acidosis, and the combined effect of pH-dependent inhibition of these channels enhances neuronal excitability and mediates CO(2) chemosensory responses in H. aspersa. We did not find a single "chemosensory channel," and the chemosensitive channels that we did find were not unique in any way that we could detect. The protein "machinery" of CO(2) chemosensitivity is probably widespread among neurons, and the selection process whereby a neuron acts or does not act as a respiratory CO(2) chemosensor probably depends on the resting membrane potential and synaptic connectivity.

Electrical properties of developing rat heart. Effects of dexamethasone.

Action potentials recorded from perinatal rat ventricles exhibited a plateau (phase 2), followed by a rapid repolarization characteristics of all mammalian ventricular cells. Within the second postnatal week, a number of distinct changes occurred in the contour of action potentials. An early slow depolarization, at the foot of the action potential, preceded the beginning of phase zero. The early slow depolarization was observed until day 12 and disappeared by day 13. A second slow depolarization occurred during the terminal phase of the rapid upstroke of the action potential, persisted through day 13 and disappeared by day 14. On day 12, what had been a homogeneous contour of action potentials seen during the first week converted into a heterogeneous contour. Occasionally, action potentials similar to those recorded from Purkinje fibres in adult heart were recorded from hearts as young as 12 days. By day 14, signs of a spike (the hallmark of action potentials from adult heart) were apparent in some fibres. Treatment of newborn rats with dexamethasone on the second day after birth prevented the disappearance of the second slow depolarization. In adult and aged rat hearts, dexamethasone treatment induced a slow depolarization and a plateau in the region of overshoot. In view of the time-dependent change of the second slow depolarization it is suggested that this phase of the action potential is influenced by the levels of circulating glucocorticoid in developing heart and by changes in calcium sensitivity observed in this species. Heterogeneity of action potentials observed on day 12 postnatal may precede structural differentiation of myofilaments.

Studies of impedance in cardiac tissue using sucrose gap and computer techniques. II. Circuit simulation of passive electrical properties and cell-to-cell transmission.

The impedance measured in a strip of heart tissue from the moth Hyalophora cecropia is fitted by circuit models of several configurations. The circuits include: (a) a single R-C circuit (b) a double R-C circuit (c) terminated transmission lines, and (d) a pattern of cells with cell-to-cell transmission paths. The last of these is found to give the best fit. Calculation of the model impedances and optimization of element values are performed by a computer. The possibility that the mechanism of cell-to-cell transmission may be capacitative rather than conductive is explored using values of capacitance derived from the circuit models to calculate the effect of capacitative coupling alone on signal transmission. The calculations show that sufficient voltage can be transmitted from the excited cell to an adjacent cell to effect excitation.

Pharmacological tests of the mechanism of the periodic rhythm caused by veratramine in the sinoatrial node of the guinea pig.

1. We investigated the effects of several drugs and extracellular ions on the periodic sinoatrial node rhythm caused by high concentrations of veratramine (>2 microM) in isolated guinea pig sinus atria. 2. During the active phase of this rhythm, pacemaker activity appeared to be due to transient afterdepolarizations resembling the delayed afterdepolarizations attributed to Ca++-induced Ca++ release in cardiac tissue. 3. Ryanodine (200-2200 nM) did not decrease the transient afterdepolarizations, and instead increased the heart rate during the active phase, prolonged the active phase, and sometimes caused conversion to regular rhythm. 4. Dichlorobenzamyl (10-110 microM), a blocker of electrogenic Na+-Ca++ exchange, did not slow or stop beating during periodic rhythm, but rather increased average heart rate and, at a higher concentration, caused conversion to regular rhythm. 5. Ouabain (0.1 microM), an inhibitor of the sodium pump and electrogenic Na+-K+ exchange, had little effect on veratramine periodic rhythm, but at higher concentrations it caused increased average heart rate and conversion to regular rhythm. 6. The chronotropic effect of Ca++ was normally weakly positive; however, in the presence of veratramine, and before the appearance of periodic rhythm, the chronotropic effect of Ca++ was weakly negative, and was associated with destabilization of the heart rate, leading to frequency oscillations or periodic rhythm. 7. Veratramine changed the chronotropic effect of K+ from weakly negative to moderately positive. 8. When half the Na+ or Cl- in the bathing medium was replaced by an impermeant ion, in the absence of veratramine the average heart rate was slightly decreased, whereas, in the presence of veratramine and periodic rhythm the average rate was increased, although the increase was not statistically significant in the case of low Na+. 9. These observations indicate that Ca++-induced Ca++ release, Na+-Ca++ exchange, and probably electrogenic Na+-K+ exchange play no important role in generation of periodic rhythm. The increased K+ dependence suggests an altered pacemaker mechanism.

Studies on the bradycardia and periodic rhythm caused by veratramine in the sinoatrial node of the guinea pig.

In spontaneously beating, isolated guinea pig sinus-atria, veratramine (2.44 microM) slowed the rate of spontaneous depolarization of sinoatrial node cells throughout diastole, markedly slowed the frequency, and often (especially in the presence of high extracellular Ca2+) induced a periodic rhythm. This rhythm consisted of periods of complete inactivity (inactive phases) alternating with periods of apparently normal beating (active phases), with a rising and falling (parabolic) frequency pattern like that of neuronal burst firing. Slight mechanical deformation of the sinoatrial node markedly attenuated the effects of veratramine, and periodic rhythm could not be produced when the sinoatrial node was pinned down for immobilization. During the active phase of periodic rhythm, the rate of spontaneous diastolic depolarization (pacemaker potential) in pacemaker cells rose and fell with the frequency, while contrariwise, the maximum rate of depolarization fell and then rose. The last beat in the active phase was followed by a small transient afterdepolarization, which had the appearance of an abortive pacemaker potential that failed to reach threshold for triggering an action potential. The effects of various programs of electrical stimulation and pacing indicated that activity of the sinoatrial node, whether spontaneous or driven, has two effects on the amplitude of afterdepolarization, a short-lasting cumulative facilitory effect and a long-lasting cumulative inhibitory effect. Veratramine periodic rhythm arises from the interplay of these two effects, with abrupt cessation of beating whenever the afterdepolarization amplitude falls below the threshold for triggering an action potential. It is suggested that the inhibitory effect may be due to inactivation of the slow inward current and the facilitory effect may be due to one or more of the depolarizing currents activated by intracellular Ca2+.

Action potentials in human macrophages are calcium spikes.

Transmembrane resting and action potentials measured with single and double microelectrode impalements of human monocyte-derived macrophages reveal that repetitive action potentials induced by depolarizing current pulses are sodium insensitive and calcium dependent. Neither amplitude, frequency of spiking, threshold nor rate of rise of the action potentials is altered by different levels of extracellular sodium, substitution of choline for sodium, or exposure to tetrodotoxin. Spiking activity is enhanced in high calcium, diminished in frequency and amplitude by lowered extracellular calcium and is blocked by cobalt and verapamil.

Ion channels in human macrophages compared with the U-937 cell line.

The human cell line U-937 has been used extensively to model many macrophage functions. We have examined the cell membranes of human monocyte-derived macrophages (HMDM) and U-937 cells to compare membrane properties as expressed by single ion channel currents. The patch-clamp technique was applied to isolated, nonactivated, inside-out patches of cell membranes obtained from HMDM and from the U-937 cell line. Voltage-gated potassium channels of similar conductance but different kinetics are present in both types of cells, and a calcium-activated potassium channel is present only in the HMDM. These differences in ion channel properties suggest fundamentally different behavior between these two cell types at the level of the cell membrane.

Barium-induced electro-mechanical uncoupling in myocardial cells.

The heart of the adult moth Hyalophora cecropia requires extracellular calcium to maintain electrogenesis as well as tension development. In this study we ask whether the processes of autorhythmicity, driven electrogenesis and tension development require calcium specifically or whether the divalent cation Ba2+ can be substituted for calcium to support these activities. Ba2+ substituted for Ca2+ in equimolar amounts caused a marked (25 mV) hyperpolarization, suppression both of pacemaker activity and of tension development in spontaneously beating semi-isolated heart cells. Heart cells bathed in Ba2+ saline and paced by action potentials (produced by external stimuli) of greatly increased amplitude, prolonged phase 2 (plateau) and increased latency, and after 30 min, no mechanical activity was observed. These changes were completely reversible when calcium was reintroduced. We conclude that Ba2+ substitution for Ca2+ is an effective electromechanical uncoupler in moth heart cells. Although Ba2+ can support electrogenesis, it cannot replace 'trigger'-Ca2+ needed to release calcium from sarcoplasmic stores to effect tension development.