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Background: Butamben or n-butyl-p-aminobenzoate is a long-acting experimental local anesthetic for the treatment of chronic pain when given as an epidural suspension. We have investigated whether Cav1.2/L-type calcium channels may be a target of this butamben action., Methods: The effect of butamben on these channels was studied in undifferentiated rat PC12-cells with the whole-cell patch-clamp technique in voltage-clamp. Ba(2+) ions were used as the charge carriers in the calcium channel currents, whereas K(+) currents were removed using K(+) free solutions., Results: Butamben 500 microM reversibly suppressed the total whole-cell barium current by 90% +/- 3% (n = 15), whereas 10 microM nifedipine suppressed this barium current by 75% +/- 7% (n = 6). Preexposure to butamben followed by washout decreased the inhibition by nifidepine to 47% +/- 5% (n = 10). These suppressive effects were not due to the measurement procedure and the drug vehicles in the solutions (<0.1% ethanol; n = 6)., Conclusions: Butamben inhibits the total barium current through expressed calcium channel types in PC12 cells, including Cav1.2/L-type channels. Because Cav1.2 channels may also occur in human nociceptive C fibers, this result allows the possibility that these L-type channels are involved in the analgesic action of butamben.

Butamben (BAB) is a local anesthetic that can be used in epidural suspensions for long-term selective suppression of dorsal root pain signal transmission and in ointments for the treatment of skin pain. Previously, high-voltage activated N-type calcium channel inhibition has been implicated in the analgesic effect of BAB. In the present study we show that low-voltage activated or T-type calcium channels may also contribute to this effect. Typical transient T-type barium currents, selectively evoked by low-voltage (-40 mV) clamp stimulation of small (approximately 20 microm diameter) dorsal root ganglion neurons from newborn mice, were inhibited by BAB with an IC50 value of approximately 200 microM. Furthermore, 200 microM BAB accelerated T-type current activation, deactivation, and inactivation kinetics, comparable to earlier observations for N-type calcium channels. Finally, 200 microM BAB had no effect on the midpoint potential and slope factor of the activation curve, although it caused a approximately 3 mV hyperpolarizing shift of the inactivation curve, without affecting the slope factor. We conclude that BAB inhibits T-type calcium channels with a mechanism associated with channel kinetics acceleration.

To contribute to the understanding of the mechanism underlying selective analgesia by epidural application of suspensions of the local anesthetic butamben (n-butyl-p-aminobenzoate; BAB), we investigated the effect of dissolved BAB on calcium channels in sensory neurons. Small-diameter dorsal root ganglion neurons from newborn mice were used to measure whole-cell barium or calcium currents through calcium channels upon voltage-clamp stimulation. BAB suppressed the voltage-step-evoked barium current of these cells in a concentration-dependent manner with a 50% inhibitory concentration of 207 +/- 14 microM (n = 40). A similar concentration dependency was found for the pharmacologically isolated N-type component of the whole-cell barium current. The time constants of inactivation and deactivation of the N-type current became smaller in the presence of BAB, thus suggesting that kinetic changes are involved in the inhibition of this current. BAB caused a similar inhibition of the total calcium current and its N-type component when these currents were evoked by command potentials with the shape of an action potential. This inhibition of calcium currents by BAB should be considered in the search for the mechanism of selective analgesia by epidural suspensions of this local anesthetic.

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.

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.

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.