Design and Synthesis of Quenched Activity-based Probes for Diacylglycerol Lipase and α,ß-Hydrolase Domain Containing Protein 6.

Diacylglycerol lipases (DAGL) are responsible for the biosynthesis of the endocannabinoid 2-arachidonoylglycerol. The fluorescent activity-based probes DH379 and HT-01 have been previously shown to label DAGLs and to cross-react with the serine hydrolase ABHD6. Here, we report the synthesis and characterization of two new quenched activity-based probes 1 and 2, the design of which was based on the structures of DH379 and HT-01, respectively. Probe 1 contains a BODIPY-FL and a 2,4-dinitroaniline moiety as a fluorophore-quencher pair, whereas probe 2 employs a Cy5-fluorophore and a cAB40-quencher. The fluorescence of both probes was quenched with relative quantum yields of 0.34 and 0.0081, respectively. The probes showed target inhibition as characterized in activity-based protein profiling assays using human cell- and mouse brain lysates, but were unfortunately not active in living cells, presumably due to limited cell permeability.

Three distinct modes of exocytosis revealed by amperometry in neuroendocrine cells.

Neurotransmission requires Ca(2+)-dependent release of secretory products through fusion pores that open and reclose (partial membrane distention) or open irreversibly (complete membrane distention). It has been challenging to distinguish between these release modes; however, in the work presented here, we were able to deduce different modes of depolarization-evoked exocytosis in neuroendocrine chromaffin and PC12 cells solely by analyzing amperometric recordings. After we determined the quantal size (Q), event half-width (t(50)), event amplitude (I(peak)), and event decay time constant (τ(decay)), we fitted scatter plots of log-transformed data with a mixture of one- and two-dimensional Gaussian distributions. Our analysis revealed three distinct and differently shaped clusters of secretory events, likely corresponding to different modes of exocytosis. Complete membrane distention, through fusion pores of widely varying conductances, accounted for 70% of the total amount of released catecholamine. Two different kinds of partial membrane distention (kiss-and-run and kiss-and-stay exocytosis), characterized by mode-specific fusion pores with unitary conductances, accounted for 20% and 10%, respectively. These results show that our novel one- and two-dimensional analysis of amperometric data reveals new release properties and enables one to distinguish at least three different modes of exocytosis solely by analyzing amperometric recordings.

Application of multiple-locus variable-number tandem-repeat analysis to determine clonal spread of toxin A-negative Clostridium difficile in a general hospital in Buenos Aires, Argentina.

Isolates from patients with Clostridium difficile infection (CDI) usually produce both toxin A (TcdA) and toxin B (TcdB), but an increasing number of reports from Europe and Asia mention infections with TcdA-negative, TcdB-positive (A-/B+) strains, usually characterized as PCR ribotype 017 (type 017). Incidence rates of CDI per 10 000 admissions in a 200-bed Argentinean general hospital were 37, 84, 67, 43, 48 and 42 for the years 2000 to 2005, respectively. The annual percentages of type 017 CDI were 7.7%, 64.6%, 91.4%, 92.0%, 75.0% and 86.4%, respectively. Comparison of 112 017-CDI patients with 41 non-017-CDI patients revealed that 017-CDI patients were more often male (68.8% vs. 46.3%; odds ratio 2.55, 95% confidence interval 1.23-5.50). All type 017 strains tested belonged to toxinotype VIII and had a 1.8-kb deletion in tcdA. In addition, 90% of tested type 017 isolates had high-level resistance to clindamycin and erythromycin, determined by the presence of the ermB gene. Multiple-locus variable-number tandem-repeat analysis (MLVA) was applied to 56 Argentinean isolates and 15 isolates from seven other countries. Country-specific clonal complexes were found in each country. Among 56 Argentinean isolates, four clonal complexes were recognized, accounting for 61% of all isolates. These clonal complexes did not show correlation over time, but seemed to be restricted to specific wards, mainly internal medicine and pulmonology wards. A total of 56% of recurrent infections were caused by a different isolate, despite identification of an identical PCR-ribotype. We conclude that C. difficile type 017 gradually replaced other circulating PCR ribotypes and that MLVA provides detailed insight into nosocomial spread.

Successful combat of an outbreak due to Clostridium difficile PCR ribotype 027 and recognition of specific risk factors.

In the period April-September 2005, an outbreak of Clostridium difficile infection (CDI) due to PCR ribotype 027 occurred among 50 patients in a 341-bed community hospital in Harderwijk, The Netherlands. A retrospective case-control study was performed to identify risk factors specific for CDI, using a group of patients with CDI (n = 45), a group of randomly selected control patients without diarrhoea (n = 90), and a group of patients with non-infectious diarrhoea (n = 109). Risk factors for CDI and for non-CDI diarrhoea were identified using multiple logistic regression analysis. Independent risk factors for CDI were: age above 65 years (OR 2.6; 95% CI 1.0-5.7), duration of hospitalization (OR 1.04 per additional day; 95% CI 1.0-1.1), and antibiotic use (OR 12.5; 95% CI 3.2-48.1). Of the antibiotics used, cephalosporins and fluoroquinolones were identified as the major risk factors for development of CDI. The risk of developing CDI was particularly high in people receiving a combination of a cephalosporin and a fluoroquinolone (OR 57.5; 95% CI 6.8-483.6). The main factors affecting the risk of non-CDI diarrhoea were proton-pump inhibitors, immunosuppressive drugs, underlying digestive system disease, previous surgery, and gastric tube feeding. The outbreak ended only after implementation of restricted use of cephalosporins and a complete ban on fluoroquinolones, in addition to general hygienic measures, cohorting of patients in a separate ward, education of staff, and intensified environmental cleaning. The results of this study support the importance of appropriate antimicrobial stewardship in the control of hospital outbreaks with C. difficile PCR ribotype 027.

Effect on diagnostic yield of repeated stool testing during outbreaks of Clostridium difficile-associated disease.

The effect on diagnostic yield of testing sequential stools was assessed during two hospital epidemics of Clostridium difficile. Using a rapid immunoassay, C. difficile-associated disease was diagnosed in 237 diarrhoeal patients, of whom 204 (86%) were diagnosed from the first faeces sample and 12 (5%) were diagnosed from follow-up samples obtained within 1 week. The remaining 21 (9%) patients yielded a positive test from stools obtained >1 week after the initial negative sample. It was concluded that repeated testing of stools for C. difficile toxin is of value in controlling outbreaks of C. difficile infection.

Use of highly discriminatory fingerprinting to analyze clusters of Clostridium difficile infection cases due to epidemic ribotype 027 strains.

We compared multilocus variable-number tandem-repeat analysis (MLVA) and macrorestriction endonuclease analysis using pulsed-field gel electrophoresis (PFGE) to determine their utility to identify clusters of Clostridium difficile infection (CDI) among 91 isolates of PCR ribotype 027 (NAP1, for North American pulsed-field type 1) from nine hospitals (and 10 general practitioners associated with one institution) in England. We also examined whether mortality in CDI cases was associated with specific MLVA subtypes. PFGE discriminated between ribotype 027 strains at >98% similarity, identifying five pulsovars (I to V) of 1 to 53 isolates. MLVA was markedly more discriminatory, identifying 23 types of 1 to 15 isolates (>71% similarity). PFGE pulsovars I and IV contained 14 and 8 MLVA types, respectively. Twenty-one of twenty-three (91%) of MLVA types were specific to individual PFGE pulsovars. Four CDI clusters were identified in institution A by conventional epidemiological analysis. MLVA typing identified two enlarged and two additional clusters. Thirty of forty-four (68%) patients in institution A with CDI caused by ribotype 027 strains were assigned to seven distinct clusters by a combination of MLVA typing and epidemiological records. Of 33 patients, comprising 14 different MLVA types, nine (27%) died by day 30 (early deaths). Eight of nine (89%) were associated with PFGE type IV C. difficile ribotype 027. Five of nine early deaths were associated with MLVA type 16, which was the dominant type in this cohort (10/33 cases); 4 other distinct MLVA types accounted for the other early deaths. MLVA was far superior to PFGE for analyzing clusters of CDI both within and between institutions. Further study is needed to examine whether subtypes of C. difficile ribotype 027 affect outcome.

Enzymatic enantioselective C-C-bond formation in microreactors.

We have demonstrated that multiple crude enzyme lysates containing a hydroxynitrile lyase can be used for the enantioselective synthesis of cyanohydrins from aldehydes in microchannels. Using a microreactor setup, two important parameters were efficiently screened consuming only minute amounts of reagents. More importantly, results from the continuous flow reaction were fully consistent with results obtained from larger batchwise processes in which a stable emulsion was formed.

Characteristics and incidence of Clostridium difficile-associated disease in The Netherlands, 2005.

During a 2-month period in 2005, 13 laboratories participated in a surveillance study of Clostridium difficile-associated disease (CDAD) in 17 hospitals in The Netherlands. The median incidence rate of CDAD was 16/10 000 patient admissions (2.2/10 000 patient-days) and varied from 1 to 46/10 000 patient admissions according to hospital. In total, 81 patients with CDAD were reported; 49 (61%) patients had nosocomial CDAD, and 29 (36%) patients were admitted to hospital when already suffering from diarrhoea. Two (2%) deaths were attributable to CDAD; both of these patients were admitted with severe community-onset CDAD and were aged >80 years. Among 64 toxinogenic isolates, ten (16%) belonged to PCR ribotype 027 and ten (16%) to PCR ribotype 014. Type 027 was identified in ten patients from one hospital during an unrecognised outbreak. Toxinotyping of the 64 isolates revealed the presence of six different toxinogenic types, with 41 (64%) isolates of toxinotype 0, ten (16%) isolates of toxinotype III, and nine (14%) isolates of toxinotype V. Of the 64 toxinogenic isolates, seven (11%) had a 39-bp deletion in the tcdC gene, 11 (17%) had an 18-bp deletion, and one (1%) had a deletion of c. 44 bp. Genes for binary toxin were present in 21 (33%) of the 64 toxinogenic isolates, mainly associated with toxinotypes III and V. It was concluded that the median CDAD incidence rate of 16/10 000 patient admissions in The Netherlands is considerably lower than that in Canada and the USA, and that the emerging type 027 can spread unnoticed. The high proportion (36%) of CDAD cases with a community onset has important implications for future studies of the epidemiology of CDAD.

Pores in synthetic nerve conduits are beneficial to regeneration.

Current opinion holds that pores in synthetic nerve guides facilitate nerve regeneration. Solid factual support for this opinion, however, is absent; most of the relevant studies assessed only morphological parameters and results have been contradictory. To evaluate the effect of pores, the rat sciatic nerve was either autografted or grafted with nonporous, macroporous (10-230 mum), and microporous (1-10 microm) biodegradable epsilon-caprolactone grafts. Twelve weeks later, the grafted nerves were resected, and the electrophysiological properties were determined in vitro. Subsequently midgraft-level sections were inspected, and peroneal nerve sections were evaluated morphometrically. Finally, the gastrocnemic and tibial muscle morphometrical properties were quantified. The microporous nerve graft performed much better than the nonporous and macroporous grafts with respect to most parameters: it was bridged by a free floating bundle that contained myelinated nerve fibers, there were more nerve fibers present distal to the graft, the electrophysiological response rate was higher, and the decrease in muscle cross-sectional area was markedly smaller. Hence, the present study demonstrates the beneficial effect of synthetic nerve guide pores on nerve regeneration, although with the caveat that not pores per se, but only small (1-10 microm) pores were effective.

Rapid diagnosis of toxinogenic Clostridium difficile in faecal samples with internally controlled real-time PCR.

A real-time PCR assay for Clostridium difficile was developed, based on the tcdB gene, which detected all known toxinogenic reference strains (n = 45), within 30 serogroups and 24 toxinotypes. The analytical sensitivity was 1 x 10(3) CFU/mL, and the detection limit in faeces was 1 x 10(5) CFU/g. The optimal protocol for DNA extraction from faecal samples involved use of the MagnaPure system with a Stool Transport and Recovery (STAR) buffer pre-treatment. In a 1-month prospective study of 85 patients with diarrhoea, the sensitivity, specificity and positive and negative predictive values of the assay were 100%, 94%, 55% and 100%, respectively, compared with the standard cell cytotoxicity assay.

Kv1.1 channels of dorsal root ganglion neurons are inhibited by n-butyl-p-aminobenzoate, a promising anesthetic for the treatment of chronic pain.

In this study, we investigated the effects of the local anesthetic n-butyl-p-aminobenzoate (BAB) on the delayed rectifier potassium current of cultured dorsal root ganglion (DRG) neurons using the patch-clamp technique. The majority of the K(+) current of small DRG neurons rapidly activates and slowly inactivates at depolarized voltages. BAB inhibited the whole-cell K(+) current of these neurons with an IC(50) value of 228 microM. Dendrotoxin K (DTX(K)), a specific inhibitor of Kv1.1, reduced the DRG K(+) current at +20 mV by 34%, consistent with an important contribution of channels incorporating the Kv1.1 subunit to the delayed rectifier current. To further investigate the mechanism of BAB inhibition, we examined its effect on Kv1.1 channels heterologously expressed in mammalian tsA201 cells. BAB inhibits the Kv1.1 channels with an IC(50) value of 238 microM, similar to what was observed for the native DRG current. BAB accelerates the opening and closing of Kv1.1, but does not alter the midpoint of steady-state activation. BAB seems to inhibit Kv1.1 by stabilizing closed conformations of the channel. Coexpression with the Kv beta 1 subunit induces rapid inactivation and reduces the BAB sensitivity of Kv1.1. Comparison of the heterologously expressed Kv1.1 and native DRG currents indicates that the Kv beta 1 subunit does not modulate the gating of the DTX(K)-sensitive Kv1.1 channels of DRG neurons. Inhibition of the delayed rectifier current of these neurons may contribute to the long-duration anesthesia attained during the epidural administration of BAB.

Urinary prostaglandin excretion in pregnancy: the effect of dietary sodium restriction.

INTRODUCTION: Dietary sodium restriction results in activation of the renin-angiotensin-aldosterone-system. In the non-pregnant situation renin release in response to a low sodium diet is mediated by prostaglandins. We studied the effect of dietary sodium restriction on urinary prostaglandin metabolism in pregnancy. PATIENTS AND METHODS: In a randomized, longitudinal study the excretion of urinary metabolites of prostacyclin (6-keto-PGF(1 alpha)and 2,3-dinor-6-keto-PGF(1 alpha)) and thromboxane A(2)(TxB(2)and 2,3-dinor-TxB(2)) was determined throughout pregnancy and post partum in 12 women on a low sodium diet and in 12 controls. RESULTS: In pregnancy the excretion of all urinary prostaglandins is increased. The 6-keto-PGF(1 alpha)/ TxB(2)-ratio as well as the 2, 3-dinor-6-keto-PGF(1 alpha)/ 2,3-dinor-TxB(2)-ratio did not significantly change in pregnancy. CONCLUISION Prostacyclin and thromboxane do not seem to play an important role in sodium balance during pregnancy.

A prospective study of speech and voice rehabilitation after total laryngectomy with the low-resistance Groningen, Nijdam and Provox voice prostheses.

In a prospective study the Groningen, Nijdam, and Provox voice prostheses were evaluated with respect to speech and voice rehabilitation. At approximately 1, 4, and 10 months after operation, patients were submitted to a standardized speaking task to evaluate phonatory skills (phrase length, phonation duration, dynamics on tone, dynamics on sentence, speech rate and availability of sound), speech quality (fluency and overall intelligibility), voice quality and stoma technique (stoma noise). Tracheoesophageal speech rehabilitation proved to be successful in 94-100% of patients, as measured at approximately 10 months after operation. Furthermore, no significant overall differences were found between the three prostheses. There was a significant improvement in time for speech rate and stoma noise. As for the time effects (e.g. improvement in performance over time) no differences between the three prostheses were found.

Effect of cAMP and ATP on the hyperpolarization-activated current in mouse dorsal root ganglion neurons.

In mouse dorsal root ganglion (DRG) neurons the activation curve of the hyperpolarization-activated current (Ih) shifted towards depolarized potentials when cAMP was present in the pipette. The relation between the midpoint potential and cAMP concentration could be described by a Hill function with a half-maximal concentration of 0.55 microM cAMP, reflecting a direct action of cAMP on the channel. With 5 mM ATP and a saturating concentration of cAMP an additional shift of the midpoint potential is observed which can be explained by phosphorylation. Application of Rp-cAMPS and Sp-cAMPS support the hypothesis of both a phosphorylation pathway and a direct effect exhibited by these molecules. The bell-shaped curves, relating the time constants for the slow and fast current components to the voltage, shifted towards positive membrane potentials when cAMP and ATP were in the pipette. The fully activated Ih/voltage relation and the reversal potential were not dependent on the presence of cAMP or ATP in the pipette. The mean resting membrane potential of -59 mV, using the perforated-patch configuration, hyperpolarized in the presence of extracellular CsCl. In the whole-cell configuration the resting membrane potential was significantly more negative at 0 microM cAMP (-61 mV) than at 100 microM cAMP (-57 mV). Thus, the activation of Ih, regulated by both the intracellular cAMP and the ATP concentration, may influence the excitability of DRG neurons.

Hyperpolarization-activated currents in the growth cone and soma of neonatal rat dorsal root ganglion neurons in culture.

Dissociated dorsal root ganglion neuron growth cones and somata from neonatal rats were voltage and current clamped with the use of the perforated-patch whole cell configuration to study the occurrence and properties of slow hyperpolarization-activated currents (Ih) at both regions. Under voltage-clamp conditions Ih, blockable by 2 mM extracellular CsCl, was present in 33% of the growth cones tested. Its steady-state activation as a function of voltage could be fitted with a single Boltzmann function with a midpoint potential of -97 mV. The time course of current activation could be best described by a double-exponential function. The magnitude of the fully activated conductance was 3.5 nS and the reversal potential amounted to -29 mV. At the soma, Ih was found in 80% of the somata tested, which is much higher than occurrence at the growth cone. The steady-state activation curve of Ih at the soma, fitted with a single Boltzmann function, had a midpoint potential of -92 mV, which was more positive than that in the growth cone. The double-exponential activation of the current was faster than in the growth cone. The fully activated conductance of 5.1 nS and the reversal potential of -27 mV were not significantly different from the values obtained at the growth cone. Membrane hyperpolarization by current-clamp pulses elicited depolarizing sags in 30% and 78% of the tested growth cones and somata, respectively, which is in agreement with our voltage-clamp findings. Termination of the hyperpolarizing current pulse evoked a transient membrane depolarization or an action potential at both sites. Application of 2 mM extracellular CsCl hyperpolarized the membrane potential reversibly by approximately 5 mV and blocked the depolarizing sags and action potentials following the current injections at these regions. Thus Ih contributes to the resting membrane potential and modulates the excitability of both the growth cone and the soma. Intracellular perfusion with the second messenger adenosine 3',5'-cyclic monophosphate (cAMP) was only possible at the soma by the use of the conventional whole cell configuration. Addition of 100 microM cAMP to the pipette solution shifted the midpoint potential of the Ih activation curve from -108 to -78 mV. The current activation time course was also accelerated. The reversal potential and the fully activated conductance underlying Ih were not changed by cAMP. These results imply that cAMP primarily affects the gating kinetics of Ih. Our results show for the first time quantitative differences in Ih properties and occurrence at the growth cone and soma membrane. These differences may reflect differences in intracellular cAMP concentration and in the expression of Ih.

The local anesthetic, n-butyl-p-aminobenzoate, reduces rat sensory neuron excitability by differential actions on fast and slow Na+ current components.

Effects of the local anesthetic, n-butyl-p-aminobenzoate, at a concentration of 100 microM, were investigated using the whole-cell voltage clamp on dorsal root ganglion neurons cultured from neonatal rat in a serum-enriched medium. During current clamp conditions, the drug either increased the firing threshold or blocked tetrodotoxin-sensitive and tetrodotoxin-resistant Na+ action potentials. These actions were reversible. Under voltage clamp conditions, inactivation of the Na+ current revealed the existence of 3 fast Na+ current components, termed F1, F2 and F3 (tetrodotoxin-sensitive) and 2 slow ones, termed S1 and S2 (tetrodotoxin-resistant). The local anesthetic shifted the midpoint potentials of Na+ inactivation curves for F1, F2 and F3 currents by 7, 21 and 6 mV, respectively, towards hyperpolarizing membrane voltages whereas it did not influence these potentials for the slow currents. The amplitudes of only F3 and S2 currents were reduced by n-butyl-p-aminobenzoate to 24 and 11%, respectively, of their control values. These results show that the local anesthetic has a differential mode of action on the 5 types of Na+ currents, which are apparently present in cultured sensory neurons. This differential action can play an important role in the selective analgesic effect observed after epidural administration of a 10% n-butyl-p-amino-benzoate suspension.

Ionic currents in cultured rat suprachiasmatic neurons.

Whole-cell voltage-clamp recordings were made from cultured neurons obtained by dissociation of the suprachiasmatic area of rat fetuses. Neurons were held for seven to 14 days in culture. These neurons possessed several voltage-dependent ionic currents. A transient inward Na+ current was present, which could be completely blocked by tetrodotoxin. No inward Ca2+ currents were detected. Three types of outward K+ currents were recorded, which could be separated to a reasonable extent by their differences in voltage sensitivity and pharmacology. These K+ currents corresponded to the transient current IA, the delayed rectifier current IKo and a calcium-dependent current IK(Ca) as described in other neurons. The A current activated at -50 mV, reached half-maximal conductance at about -30 mV and maximum conductance between 0 and 30 mV. During depolarizing steps it inactivated completely within 100 ms and steady-state inactivation was half-maximal at -66 mV. The outward rectifier activated at -30 mV, reached half-maximal conductance close to 0 mV and maximum conductance at about 70 mV. Slow inactivation of IKo occurred with 50% reduction in amplitude at the end of 2 s depolarizations above 0 mV. The K+ channel blocker 4-amino-pyridine (4 mM) reduced the amplitude of IA by 21% and of IKo by 32%, whereas tetraethylammonium (10 mM) decreased IA by 27% and IKo by 83%. The calcium-dependent K+ component was also voltage dependent and was present at voltages more positive than 0 mV. No inward rectifying K+ current was present. Considering its voltage dependence, IA must play a role in determining the excitability of these neurons, through its probable influence on the action potential threshold and interspike interval. Both IA and IKo should take part in membrane repolarization following an action potential. The Ca(2+)-dependent current should also contribute to repolarization following any event which gives rise to an increase in intracellular Ca2+. Apart from IA, which may make a slight contribution, none of these currents appear to be involved in determining the resting membrane potential. All three outward current components will act together in suprachiasmatic neurons to control their spontaneous firing frequency, which is the major feature of the output of these neurons in vivo. Variations in properties of these conductances could contribute to the circadian rhythm in firing frequency described in suprachiasmatic hypothalamic neurons.

Acquired neuromyotonia: evidence for autoantibodies directed against K+ channels of peripheral nerves.

Acquired neuromyotonia is characterized by hyperexcitability of motor nerves leading to muscle twitching, cramps, and weakness. The symptoms may improve following plasma exchange, and injection of immunoglobulin G (IgG) from 1 neuromyotonia patient into mice increased the resistance of neuromuscular transmission to d-tubocurarine. Here we examine nerves and muscle in vitro from mice injected with plasma or purified IgG from 6 neuromyotonia patients or pooled control subjects, and cultured dorsal root ganglion cells after treatment with IgG. Three of the patients had antibodies against human voltage-gated potassium channels labeled with 125I-alpha-dendrotoxin. The quantal release of acetylcholine (quantal content) at end-plates in diaphragms from mice treated with neuromyotonia IgG preparations was increased by 21% relative to control values (p = 0.0053). With one IgG preparation, the duration of the superficial peroneal nerve compound action currents was increased by 93%. The dorsal root ganglion cells treated with this IgG showed a marked increase in repetitive firing of action potentials. All effects were similar to those obtained with aminopyridines. We conclude that at least some patients with acquired neuromyotonia have antibodies directed against aminopyridine- or alpha-dendrotoxin-sensitive K+ channels in motor and sensory neurons, and they are likely to be implicated in the disease process.

The local anesthetic n-butyl-p-aminobenzoate selectively affects inactivation of fast sodium currents in cultured rat sensory neurons.

BACKGROUND: Aqueous suspensions of the local anesthetic n-butyl-p-aminobenzoate (BAB), epidurally applied in terminal cancer patients, resulted in a sensory blockade, lasting up to several months. To investigate the mechanism of action on the cellular level, the effect of 100 microM BAB on Na+ action potentials and on Na+ currents in dorsal root ganglion neurons from neonatal rats was studied. METHODS: Small neurons grown in cell culture were selected for patch-clamp measurements. Both Na+ action potentials, evoked by current pulses of increasing amplitude (current clamp) and Na+ currents, activated at different membrane potentials (voltage clamp), were investigated in the absence and presence of 100 microM BAB. The local anesthetic was applied by external perfusion for 2 or 10 min. RESULTS: In the presence of 100 microM BAB, either the firing threshold was raised or the action potential was abolished. The maximal peak conductances, underlying the fast sodium current INa,F and the slow sodium current INa,5, were not changed. However, the inactivation of INa,F was increased by BAB. The sigmoid inactivation curve shifted 12 mV toward hyperpolarizing membrane voltages, whereas no changes were found for the inactivation of the slow Na+ current. Only at short exposure times of 2 min, the effects of BAB could be reversed during a 10-min wash-out. CONCLUSIONS: BAB dramatically increased the firing threshold, and in part of the sensory neurons, it blocked the action potential. The inactivation of the fast Na+ channels, but not of the slow Na+ channels, was increased by BAB. Thus, the block of fast Na+ channels by BAB may contribute to epidural analgesia. At exposure times of 10 min, the effect of BAB was not reversible. This probably originates from its high lipid-solubility, which may be an important factor in determining the duration of the block in vivo.