A beginner's guide to computer simulation of voltage-gated ion conductances.

This article provides a simple introduction to the simulation of voltage-dependent ion conductances in both macroscopic and single-channel modes. Only Markovian (time-independent) systems are considered. The programmes listed are written in Microsoft QBasic or QuickBASIC but versions in other languages are available. The Hodgkin-Huxley Na+ current is used as a starting system for which an explicit macroscopic solution may be obtained and compared with the results of numerical simulations employing 4th order Runge-Kutta integration. Non-Hodgkin-Huxley behaviour such as voltage-independent inactivation and double exponential current decay are discussed and simulated. A stochastic programme is used to simulate single channel behaviour. The problems and methodologies involved in fitting experimental data using complex kinetic schemes are briefly discussed, as are alternative sources of simulation software.

Local anesthetics potently block a potential insensitive potassium channel in myelinated nerve.

Effects of some local anesthetics were studied in patch clamp experiments on enzymatically demyelinated peripheral amphibian nerve fibers. Micromolar concentrations of external bupivacaine depolarized the excised membrane considerably. The flicker K+ channel was found to be the most sensitive ion channel to local anesthetics in this preparation. Half-maximum inhibiting concentrations (IC50) for extracellular application of bupivacaine, ropivacaine, etidocaine, mepivacaine, lidocaine, and QX-314 were 0.21, 4.2, 8.6, 56, 220, and > 10,000 microM, respectively. The corresponding concentration-effect curves could be fitted under the assumption of a 1:1 reaction. Application from the axoplasmic side resulted in clearly lower potencies with IC50 values of 2.1, 6.6, 16, 300, 1,200, and 1,250 microM, respectively. The log(IC50)-values of the local anesthetics linearly depended on the logarithm of their octanol:buffer distribution coefficients with two regression lines for the piperidine derivatives and the standard amino-amides indicating an inherently higher potency of the cyclic piperidine series. Amide-linked local anesthetics did not impair the amplitude of the single-channel current but prolonged the time of the channel to be in the closed state derived as time constants tau c from closed-time histograms. With etidocaine and lidocaine tau c was 133 and 7.2 ms, and proved to be independent of concentration. With the most potent bupivacaine time constants of wash in and wash out were 1.8 and 5.2 s for 600 nM bupivacaine. After lowering the extracellular pH from 7.4 to 6.6, externally applied bupivacaine showed a reduced potency, whereas at higher pH of 8.2 the block was slightly enhanced. Intracellular pH of 6.4, 7.2, 8.0 had almost no effect on internal bupivacaine block. It is concluded that local anesthetics block the flicker K+ channel by impeding its gating but not its conductance. The slow blocker bupivacaine and the fast blocker lidocaine compete for the same receptor. Lipophilic interactions are of importance for blockade but besides a hydrophobic pathway, there exists also a hydrophilic pathway to the binding site which could only be reached from the cytoplasmic side of the membrane. Under physiological conditions, blockade of the flicker K+ channel which is more sensitive to bupivacaine than the Na+ channel might lead via membrane depolarization and the resulting sodium channel inactivation to a pronounced block of conduction in thin fibers.

Two cases of acute methanol poisoning partially treated by oral 4-methylpyrazole.

OBJECTIVE: Since the use of 4-methylpyrazole (4-MP) in the treatment of humans with methanol poisoning is poorly documented, we report two cases of acute methanol intoxication partially treated by this potent alcohol dehydrogenase (ADH) inhibitor. SETTING: Intensive Care Unit in a university hospital. PATIENTS: A 56-year-old man and an 18-year-old woman were observed, respectively, 41 and 16 h after the voluntary ingestion of an unknown amount of methanol. INTERVENTION: In both cases, ethanol was used as the first antidote. In the first patient, hemodialysis was also performed on admission because a high methanol level (0.72 g/l) and visual impairment were noted. In the second patient, ethanol therapy was withdrawn after 12 h when clinical and biological signs of acute pancreatitis became evident. Both patients received multiple oral doses of 4-MP. No recurrence of metabolic acidosis occurred and the 4-MP therapy was well tolerated. CONCLUSION: While the use of 4-MP is better documented in cases of ethylene glycol poisoning, it could also become an accepted option for the management of methanol poisoning since 4-MP offers advantages over ethanol therapy.

[Pain treatment tomorrow]. / Schmerztherapie morgen.

In the last few years global understanding of pain has improved due to current molecular biological studies. The identification of a large number of different proteins is an essential part of future therapies, since they, as enzymes, receptors or ion channels, are specific relays in the nociceptive system and therefore have key functions in pharmacotherapeutic therapy. Nature itself supplies a variety of substances which are of therapeutic value, some of which are already in scientific trial. In contrast, even today not all available pain therapy measures are in use in Germany, at least not in all areas. Especially the treatment of children is not state of the art. Thus a further increase of chronic pain patients is to be expected in the future. This development has to be stopped, not only for ethical reasons, but also to prove the economical value of adequate pain therapy. Qualified treatment of acute pain within well defined limits can help to avoid chronification and further costs. This will be the decisive argument with which to mobilize the necessary funds for the development of the pain treatment of the future.

Local anaesthetic effects on tetrodotoxin-resistant Na+ currents in rat dorsal root ganglion neurones.

Besides the fast tetrodotoxin-sensitive Na+ current, small dorsal root ganglion neurones of rats also possess a slower tetrodotoxin-resistant Na+ current. The blocking effect of commonly used local anaesthetics upon the tetrodotoxin-resistant Na+ current was investigated in the present paper. Dorsal root ganglia were dissected from adult rats and cells were enzymatically isolated. The whole-cell patch clamp technique was then used to measure inward Na+ currents of small dorsal root ganglion neurones. Externally applied local anaesthetics reversibly blocked the tetrodotoxin-resistant Na+ current in a dose-dependent manner. Half-maximal blocking concentrations for tonic block were: lignocaine, 326 microM; prilocaine, 253 microM; mepivacaine, 166 microM; etidocaine, 196 microM bupivacaine, 57 microM procaine, 518 microM benzocaine, 489 microM; tetracaine, 21 microM; and dibucaine, 23 microM. Blocking of the current by lignocaine was independent of temperature. The quaternary lignocaine derivative OX-314 did not have any effect upon the tetrodotoxin-resistant Na+ current when applied externally. High concentrations of tetrodotoxin also blocked the tetrodotoxin-resistant Na+ current with a half-maximal blocking concentration of 115 microM. The block by high tetrodotoxin concentrations did not compete with the lignocaine block, suggesting that there were two independent blocking mechanisms for the two substances. The tetrodotoxin-resistant Na+ currents also showed a marked sensitivity to phasic (use-dependent) block by local anaesthetics.

Stereoselectivity of bupivacaine in local anesthetic-sensitive ion channels of peripheral nerve.

BACKGROUND: The local anesthetic bupivacaine exists in two stereoisomeric forms, R(+)- and S(-)-bupivacaine. Because of its lower cardiac and central nervous system toxicity, attempts were made recently to introduce S(-)-bupivacaine into clinical anesthesia. We investigated stereoselective actions of R(+)-and S(-)-bupivacaine toward two local anesthetic-sensitive ion channels in peripheral nerve, the Na+ and the flicker K+ channel. METHODS: In patch-clamp experiments on enzymatically demyelinated peripheral amphibian nerve fibers, Na+ and flicker K+ channels were investigated in outside-out patches. Half-maximum inhibiting concentrations (IC50) were determined. For the flicker K+ channel, simultaneous block by R(+)-bupivacaine and S(-)-bupivacaine was analyzed for competition and association (k1) and dissociation rate constants (k(-1)) were determined. RESULTS: Both channels were reversibly blocked by R(+)- and S(-)-bupivacaine. The IC50 values (+/- SEM) for tonic Na+ channel block were 29+/-3 microM and 44+/-3 microM, respectively. IC50 values for flicker K+ channel block were 0.15+/-0.02 microM and 11+/-1 microM, respectively, resulting in a high stereopotency ratio (+/-) of 73. Simultaneously applied enantiomers competed for a single binding site. Rate constants k1 and k(-1) were 0.83+/-0.13x10(6) M(-1) x S(-1) and 0.13+/-0.03 s(-1), respectively, for R(+)-bupivacaine and 1.90+/-0.20x10(6) M(-1) x s(-1) and 8.3+/-1.0 s(-1), respectively, for S(-)-bupivacaine. CONCLUSIONS: Bupivacaine block of Na+ channels shows no salient stereoselectivity. Block of flicker K+ channels has the highest stereoselectivity ratio of bupivacaine action known so far. This stereoselectivity derives predominantly from a difference in k(-1), suggesting a tight fit between R(+)-bupivacaine and the binding site. The flicker K+ channel may play an important role in yet unknown toxic mechanisms of R(+)-bupivacaine.

Simultaneous detection and quantification of acidic and basic drugs in serum toxicological screening: use of ion-pairing technique in liquid-liquid extraction.

The toxicological screening, using the combination of high performance liquid chromatography with diode array ultraviolet detector and ion-pairing technique in liquid-liquid extraction, is an effective tool in the identification and quantification of the acidic and basic substances in a single run. The use of an ion-pairing technique in the conventional extraction shows the co-extraction of the uncharged and charged form of the analytes present in a serum sample. The stationary phase used is C-18-bonded phase. The mobile phase is acetonitrile--phosphate buffer (pH 3; 25 mM) containing 25 mM triethylammonium as ion-pairing agent. The analytical validation shows reproducible recoveries, good day-to-day repeatability and sensible results compatible with clinical and forensic use.

[Possible applications of the "patch-clamp" method in anesthesiologic research; comment]. / Einsatzmöglichkeiten der "Patch-Clamp"-Methode in der anästhesiologischen Forschung.

The patch clamp technique has gained considerable significance during the past two decades since it was developed. Many mechanisms of cell function have been elucidated by the application of the patch clamp method because it was now possible to demonstrate and investigate directly single ion channels in excitable cell membranes. An important discovery, as far as medicine was concerned, was that various clinically used substances interact directly with ion channels. Local anaesthetics, antiarrhythmics, antidiabetics, muscle relaxants are a few examples. For these reasons, more and more clinical physicians use the patch clamp method for their specific investigations. Collaboration between a clinical institute and a basic research department can often be found, which gives clinical research more background and basic science more practical aspects.

Blocking mechanisms of ketamine and its enantiomers in enzymatically demyelinated peripheral nerve as revealed by single-channel experiments.

BACKGROUND: Ketamine shows, besides its general anesthetic effect, a local anesthetic-like action that is due to blocking of peripheral nerve sodium currents. In this study, the stereoselectivity of the blocking effects of the ketamine enantiomers S(+) and R(-) was investigated in sodium and potassium channels in peripheral nerve membranes. METHODS: Ion channel blockade of ketamine was investigated in enzymatically dissociated Xenopus sciatic nerves in multiple-channel and in single-channel outside-out patches. RESULTS: Concentration-effect curves for the Na+ peak current revealed half-maximal inhibiting concentrations (IC50) of 347 microM and 291 microM for S(+) and R(-) ketamine, respectively. The potential-dependent K+ current was less sensitive than the Na+ current with IC50 values of 982 microM and 942 microM. The most sensitive ion channel was the flickering background K+ channel, with IC50 values of 168 microM and 146 microM for S(+) and R(-) ketamine. Competition experiments suggest one binding site at the flicker K+ channel, with specific binding affinities for each of the enantiomers. For the Na+ channel, the block was weaker in acidic (pH = 6.6) than in neutral (pH = 7.4) and basic (pH = 8.2) solutions; for the flicker K+ channel, the block was weaker in acidic and stronger in basic solutions. CONCLUSIONS: Ketamine blockade of sodium and potassium channels in peripheral nerve membranes shows no stereoselectivity except for the flicker K+ channel, which showed a very weak stereoselectivity in favor of the R(-) form. This potential-insensitive flicker K+ channel may contribute to the resting potential. Block of this channel and subsequent depolarization of the resting membrane potential leads, besides to direct Na+ channel block, to inexcitability via Na+ channel inactivation.

Single-channel recording in myelinated nerve fibers reveals one type of Na channel but different K channels.

Amphibian myelinated nerve fibers were treated with collagenase and protease. Axons with retraction of the myelin sheath were patch-clamped in the nodal and paranodal region. One type of Na channel was found. It has a single-channel conductance of 11 pS (15 degrees C) and is blocked by tetrodotoxin. Averaged events show the typical activation and inactivation kinetics of macroscopic Na current. Three potential-dependent K channels were identified (I, F, and S channel). The I channel, being the most frequent type, has a single-channel conductance of 23 pS (inward current, 105 mM K on both sides of the membrane), activates between -60 and -30 mV, deactivates with intermediate kinetics, and is sensitive to dendrotoxin. The F channel has a conductance of 30 pS, activates between -40 and 60 mV, and deactivates with fast kinetics. The former inactivates within tens of seconds; the latter inactivates within seconds. The third type, the S channel, has a conductance of 7 pS and deactivates slowly. All three channels can be blocked by external tetraethylammonium chloride. We suggest that these distinct K channel types form the basis for the different components of macroscopic K current described previously.

Fundamental properties of local anesthetics: half-maximal blocking concentrations for tonic block of Na+ and K+ channels in peripheral nerve.

UNLABELLED: Local anesthetics suppress excitability by interfering with ion channel function. Ensheathment of peripheral nerve fibers, however, impedes diffusion of drugs to the ion channels and may influence the evaluation of local anesthetic potencies. Investigating ion channels in excised membrane patches avoids these diffusion barriers. We investigated the effect of local anesthetics with voltage-dependent Na+ and K+ channels in enzymatically dissociated sciatic nerve fibers of Xenopus laevis using the patch clamp method. The outside-out configuration was chosen to apply drugs to the external face of the membrane. Local anesthetics reversibly blocked the transient Na+ inward current, as well as the steady-state K+ outward current. Half-maximal tonic inhibiting concentrations (IC50), as obtained from concentration-effect curves for Na+ current block were: tetracaine 0.7 microM, etidocaine 18 microM, bupivacaine 27 microM, procaine 60 microM, mepivacaine 149 microM, and lidocaine 204 microM. The values for voltage-dependent K+ current block were: bupivacaine 92 microM, etidocaine 176 microM, tetracaine 946 microM, lidocaine 1118 microM, mepivacaine 2305 microM, and procaine 6302 microM. Correlation of potencies with octanol:buffer partition coefficients (logP0) revealed that ester-bound local anesthetics were more potent in blocking Na+ channels than amide drugs. Within these groups, lipophilicity governed local anesthetic potency. We conclude that local anesthetic action on peripheral nerve ion channels is mediated via lipophilic drug-channel interactions. IMPLICATIONS: Half-maximal blocking concentrations of commonly used local anesthetics for Na+ and K+ channel block were determined on small membrane patches of peripheral nerve fibers. Because drugs can directly diffuse to the ion channel in this model, these data result from direct interactions of the drugs with ion channels.

A TEA-insensitive flickering potassium channel active around the resting potential in myelinated nerve.

A novel potassium-selective channel which is active at membrane potentials between -100 mV and +40 mV has been identified in peripheral myelinated axons of Xenopus laevis using the patch-clamp technique. At negative potentials with 105 mM-K on both sides of the membrane, the channel at 1 kHz resolution showed a series of brief openings and closings interrupted by longer closings, resulting in a flickery bursting activity. Measurements with resolution up to 10 kHz revealed a single-channel conductance of 49 pS with 105 mM-K and 17 pS with 2.5 mM-K on the outer side of the membrane. The channel was selective for K ions over Na ions (PNa/PK = 0.033). The probability of being within a burst in outside-out patches varied from patch to patch (> 0.2, but often > 0.9), and was independent of membrane potential. Open-time histograms were satisfactorily described with a single exponential (tau o = 0.09 msec), closed times with the sum of three exponentials (tau c = 0.13, 5.9, and 36.6 msec). Sensitivity to external tetraethylammonium was comparatively low (IC50 = 19.0 mM). External Cs ions reduced the apparent unitary conductance for inward currents at Em = -90 mV (IC50 = 1.1 mM). Ba and, more potently, Zn ions lowered not only the apparent single-channel conductance but also open probability. The local anesthetic bupivacaine with high potency reduced probability of being within a burst (IC50 = 165 nM). The flickering K channel is clearly different from the other five types of K channels identified so far in the same preparation. We suggest that this channel may form the molecular basis of the resting potential in vertebrate myelinated axons.

Differential block of fast and slow inactivating tetrodotoxin-sensitive sodium channels by droperidol in spinal dorsal horn neurons.

BACKGROUND: Dorsal horn neurons of the spinal cord participate in neuronal pain transmission. During spinal and epidural anesthesia, dorsal horn neurons are exposed to local anesthetics and opioids. Droperidol is usually given with opioids to avoid nausea and vomiting. A recently developed method of "entire soma isolation" has made it possible to study directly the action of droperidol on different components of Na+ current in dorsal horn neurons. METHODS: Using a combination of the whole-cell patch-clamp recording from spinal cord slices and the entire soma isolation method, we studied the direct action of droperidol on two types of Na+ currents in dorsal horn neurons of young rats. RESULTS: The tetrodotoxin-sensitive Na+ current in isolated somata consisted of a fast inactivating (tauF, 0.5-2 ms; 80-90% of the total amplitude) and a slow inactivating (tauS, 6-20 ms; 10-20% of the total amplitude) component. Droperidol, at concentrations relevant for spinal and epidural anesthesia, selectively and reversibly suppressed the fast component with a half-maximum inhibiting concentration (IC50) of 8.3 microm. The slow inactivating component was much less sensitive to droperidol; the estimated IC50 value was 809 microm. CONCLUSIONS: Droperidol selectively blocks fast Na+ channels, the fast and slow components of the Na+ current in dorsal horn neurons are carried through pharmacologically distinct types of Na+ channels, and the effects of droperidol differ from those of local anesthetics and tetrodotoxin, which equipotently suppress both components. Droperidol may be suggested as a pharmacologic tool for separation of different types of inactivating tetrodotoxin-sensitive Na+ channel.

[Partial CO(2) rebreathing technique versus thermodilution: measurement of cardiac output before and after operations with extracorporeal circulation]. / "Partielle CO(2)-Rückatmungstechnik" versus Thermodilution Bestimmung des Herzzeitvolumens vor und nach Eingriffen mit extrakorporaler Zirkulation.

BACKGROUND: The NICO(2) monitor determines "pulmonary capillary blood flow" (Qpc) and cardiac output (Qt) using the "partial CO(2) rebreathing technique". The agreement between NICO(2) and thermodilution (TD) cardiac output was compared before and after cardiac surgery with cardiopulmonary bypass (CBP). In addition, the possibility of calculating the intrapulmonary shunt fraction (Qs/Qt) by combining data from the NICO(2) monitor and the TD was investigated. METHODS: In 32 patients measurements were made following induction of anesthesia ("pre-CBP"), 30 min after weaning from CBP ("post-CBP"), and 6-8 h after surgery ("post-OP"). Qt was determined by the NICO(2) monitor and TD, Qpc by the NICO(2) monitor, and Qs/Qt(O(2)) from the standard formula. An intrapulmonary shunt was calculated using Qpc(NICO(2)) and Qt(TD) according to the equation Qs/Qt=1-Qpc/Qt. Bland-Altman and regression analysis techniques were used for statistical evaluation. RESULTS: "Pre-CBP" there was a good agreement between Qt(NICO(2)) and Qt(TD) with both a bias and precision of -0.13+/-0.46 l/min and a correlation of r=0.88+/-0.47 ( p<0.001). In contrast, "post-CBP" and "post-OP" there was a lack of agreement for Qt (bias and precision: 0.97+/-1.05 l/min and -0.33+/-0.8 l/min, respectively). Regarding the shunt calculations no significant correlations between methods could be found. CONCLUSION: Cardiac output measurement by the NICO(2) monitor agree well with TD under steady-state conditions but after CBP the agreement was too small. Combining Qpc(NICO(2)) and Qt(TD) does not offer a reliable possibility for calculating intrapulmonary shunt.

Effect of bupivacaine on ATP-dependent potassium channels in rat cardiomyocytes.

Bupivacaine induces fatal arrhythmia when accidentally injected i.v. or overdosed, whereas lidocaine is used as an anti-arrhythmic agent. We have suggested recently that the anti-arrhythmic effect of lidocaine may be explained by suppression of ATP-sensitive potassium (KATP) channels. Therefore, it could be argued that different sensitivities of KATP channels to both drugs could be a reason for their different arrhythmic and anti-arrhythmic properties. In this study, we have investigated the direct action of bupivacaine on KATP channels in cardiomyocytes. The effects of bupivacaine on the cardiac KATP channel were investigated using the patch-clamp technique on enzymatically dissociated cardiomyocytes of adult rats. Bupivacaine was applied to the outer side of excised membrane patches using a multiple-barrel perfusion system. Concentration-response curves indicated that bupivacaine blocked the mean current of the KATP channels at a half-maximum inhibiting concentration (IC50) of 29 mumol litre-1, similar to that reported for lidocaine (43 mumol litre-1). Binding of bupivacaine influenced the gating of this channel, but did not reduce the conductance of the open channel. Bupivacaine and lidocaine were equipotent in blocking KATP channels. However, because of its excessive block of the sodium channel in the inactivated state, block of KATP channels by bupivacaine will only enhance its cardiotoxicity.

Electrophysiological properties of sodium current subtypes in small cells from adult rat dorsal root ganglia.

1. Whole-cell and single-channel Na+ currents were recorded from small (ca. 20 micron diameter) cells isolated from adult rat dorsal root ganglia (DRG). Currents were classified by their sensitivity to 0.3 microM tetrodotoxin (TTX), electrophysiological properties and single-channel amplitude. Cells were classified according to the types of current recorded from them. 2. Type A cells expressed essentially pure TTX-sensitive (TTX-S) currents. Availability experiments with prepulse durations between 50 ms and 1 s gave a half-available voltage (Vh) of around -65 mV but the availability curves often had a complex shape, consistent with multiple inactivation processes. Measured inactivation time constants ranged from less than 1 ms to over 100 s, depending on the protocol used. 3. Cell types B and C each had, in addition to TTX-S currents, substantial and different TTX-resistant (TTX-R) currents that we have designated TTX-R1 and TTX-R2, respectively. TTX-R1 currents had a 1 s Vh of -29 mV, showed little 1 Hz use dependence at -67 mV and recovered from the inactivation induced by a 60 ms depolarizing pulse with time constants of 1.6 ms (91 %) and 908 ms. They also exhibited slow inactivation processes with component time constants around 10 and 100 s. TTX-R2 currents activated and inactivated at more negative potentials (1 s Vh = -46 mV), showed substantial 1 Hz use dependence and had inactivation (60 ms pulse) recovery time constants at -67 mV of 3.3 ms (58 %) and 902 ms. 4. Type D cells had little or no current in 0.3 microM TTX at a holding potential of -67 mV. Current amplitude increased on changing the holding potential to -107 mV. Type D cell currents had more hyperpolarized availability and I-V curves than even TTX-R2 currents and suggest the existence of TTX-R3 channels. 5. In outside-out patches with 250 mM external NaCl, the single-channel conductance (gamma) of TTX-S channels was 19.5 pS and the potential for half-maximal activation (Va) was -45 mV. One population of TTX-R channels had a gamma of 9.2 pS and a Va of -27 mV. A second population had a gamma of 16.5 pS and a more negative Va of -42 mV. The latter population may underlie the type D cell current. 6. Small DRG cells express multiple Na+ currents with varied time constants and voltage dependences of activation and inactivation. Nociceptive cells still fire when chronically depolarized by an increased external K+ concentration. TTX-R1 and TTX-R2 Na+ channels may support that firing, while the range of inactivation time constants described here would increase the repertoire of DRG cell burst firing behaviour generally.

Splicing in a plant pararetrovirus.

Analysis of expression in rice cells of plasmids in which an ATG-less chloramphenicol acetyl transferase ORF was placed in frame with the coding sequence of the pararetrovirus rice tungro bacilliform virus (RTBV) ORF IV, and which contained much of the upstream full-length transcript sequence of RTBV, gave evidence to suggest that a splicing event occurred. Reverse transcription/PCR of RNA from transfected protoplasts and from RTBV-infected plants yielded a product which confirmed that the 5' end of ORF IV was spliced in frame to a short ORF (sORF) in the RTBV leader sequence removing an intron of about 6.3 kb. Most of the translation is initiated at the ATG codon in the sORF with only about 10% at the ORF IV ATG codon. The efficiency of splicing appears to be inversely related to the length of the intron. The finding of splicing in a pararetrovirus blurs the differences between them and retroviruses but is in accord with the hypothesis that retroelements acquire genes and sequences which adapt them to specific niches.

Basic electrical properties of in situ endothelial cells of small pulmonary arteries during postnatal development.

Small pulmonary arteries are the major determinants of pulmonary artery pressure and vascular resistance. Their endothelium modulates pulmonary resistance, remodeling, and blood fluidity. We developed a method that provides access to the luminal surface of small pulmonary arteries of rat and allows the patch-clamp study of electrical properties of in situ endothelium. At birth, the membrane was predominantly permeable for K(+), showing a resting potential of -70 mV. This conductance was not voltage-dependent and was insensitive to standard blockers of K(+) channels such as tetraethylammonium, charybdotoxin, and 4-aminopyridine. The first 22 d of development were accompanied by an additional expression of a Cl(-) conductance, increasing membrane potential to -45 mV. Acidosis reduced K(+) conductance and depolarized the membrane, whereas alkalosis resulted in hyperpolarization. Two-electrode recordings revealed tight electrical coupling (83%) between neighboring cells in the circumferential direction of the artery. The electrotonic length constant for endothelium was 13.3 microm, indicating that most cells in one cross section of a small artery are well coupled. Thus, the resting membrane conductances in small pulmonary artery endothelial cells change with postnatal development and are modulated by pH.

Effect of drugs used for neuropathic pain management on tetrodotoxin-resistant Na(+) currents in rat sensory neurons.

BACKGROUND: Tetrodotoxin-resistant Na(+) channels play an important role in generation and conduction of nociceptive discharges in peripheral endings of small-diameter axons of the peripheral nervous system. Pathophysiologically, these channels may produce ectopic discharges in damaged nociceptive fibers, leading to neuropathic pain syndromes. Systemically applied Na(+) channel--blocking drugs can alleviate pain, the mechanism of which is rather unresolved. The authors investigated the effects of some commonly used drugs, i.e., lidocaine, mexiletine, carbamazepine, amitriptyline, memantine, and gabapentin, on tetrodotoxin-resistant Na+ channels in rat dorsal root ganglia. METHODS: Tetrodotoxin-resistant Na(+) currents were recorded in the whole-cell configuration of the patch-clamp method in enzymatically dissociated dorsal root ganglion neurons of adult rats. Half-maximal blocking concentrations were derived from concentration-inhibition curves at different holding potentials (-90, -70, and -60 mV). RESULTS: Lidocaine, mexiletine, and amitriptyline reversibly blocked tetrodotoxin-resistant Na(+) currents in a concentration- and use-dependent manner. Block by carbamazepine and memantine was not use-dependent at 2 Hz. Gabapentin had no effect at concentrations of up to 3 mm. Depolarizing the membrane potential from -90 mV to -60 mV reduced the available Na(+) current only by 23% but increased the sensitivity of the channels to the use-dependent blockers approximately fivefold. The availability curve of the current was shifted by 5.3 mV to the left in 300 microm lidocaine. CONCLUSIONS: Less negative membrane potential and repetitive firing have little effect on tetrodotoxin-resistant Na(+) current amplitude but increase their sensitivity to lidocaine, mexiletine, and amitriptyline so that concentrations after intravenous administration of these drugs can impair channel function. This may explain alleviation from pain by reducing firing frequency in ectopic sites without depressing central nervous or cardiac excitability.

ATP-dependent potassium channel in rat cardiomyocytes is blocked by lidocaine. Possible impact on the antiarrhythmic action of lidocaine.

BACKGROUND: During myocardial ischemia, lidocaine has favorable antiarrhythmic properties. Malignant arrhythmias result from heterogeneity between ischemic and nonischemic regions in extracellular potassium concentration and action potential duration. These effects have been attributed to the activation of ATP-dependent potassium (KATP) channels. In this study, we investigated the action of lidocaine on the KATP channels to test the possible link between the antiarrhythmic properties of lidocaine and its action on the KATP channel. METHODS AND RESULTS: The patch-clamp technique was employed on enzymatic dissociated cardiomyocytes of adult rats. Lidocaine was applied to the outer side of excised membrane patches by means of a multibarrel perfusion system. Lidocaine reversibly blocked the mean current of the KATP channels in a concentration-dependent manner (IC50 = 43 +/- 4.7 mumol/L, E = 0 mV, n = 6), while the amplitude of the single-channel current remained unchanged. The half-maximum blocking concentration corresponds to the therapeutic range for the antiarrhythmic application of a lidocaine bolus in humans. CONCLUSIONS: The open probability but not the conductance of the KATP channel in the membrane of rat cardiomyocytes is blocked by lidocaine. This action may explain, in part, the favorable antiarrhythmic properties of lidocaine during acute myocardial ischemia.