Francisco Walber Ferreira-da-Silva

Linalool blocks excitability in peripheral nerves and voltage-dependent Na+ current in dissociated dorsal root ganglia neurons

Linalool is a terpene that occurs as a major constituent of essential oils of many plants of widespread distribution. It possesses several biological and pharmacological activities, including depressant effects on the central nervous system and olfactory receptors. The present study investigated whether linalool affects the excitability of peripheral components of the somatic sensory system. We used sciatic nerve and preparations of intact and dissociated neurons of dorsal root ganglion for extracellular, intracellular and patch-clamp recordings. Linalool concentration-dependently (0.3-2.0mM) and reversibly blocked the excitability of the sciatic nerve. It inhibited peak-to-peak amplitude of the compound action potential (IC(50) was 0.78+/-0.04 mM). At 0.8mM, it reversibly increased rheobase and chronaxy (from 3.2+/-0.1 V and 52.4+/-4.1 micros to 4.2+/-0.3 V and 71.2+/-5.5 micros (n=5), respectively) and inhibited with greater pharmacological potency the amplitude of the compound action potential components corresponding to axons with slower velocity of conduction. In a similar concentration range (0.1-6mM), linalool concentration-dependently and reversibly blocked the generation of action potentials of intact dorsal root ganglion neurons without alteration of resting membrane potential and input resistance, and inhibited the voltage-gated Na(+) current of dissociated dorsal root ganglion neurons. In conclusion, we demonstrated that linalool acts on the somatic sensory system with local anesthetic properties, since it blocked the action potential by acting on voltage-dependent Na(+) channels. This finding is important in showing the potential usefulness of linalool as a pharmacotherapeutic agent.

Effects of 1,8-cineole on electrophysiological parameters of neurons of the rat superior cervical ganglion.

1  1,8‐Cineole is a non‐toxic small terpenoid oxide believed to have medicinal properties in folk medicine. It has been shown to have various pharmacological effects, including blockade of the compound action potential (AP). In the present study, using intracellular recording techniques, we investigated the effects of 1,8‐cineole on the electrophysiological parameters of neurons of the superior cervical ganglion (SCG) in rats. 2  1,8‐Cineole (0.1–6 mmol/L) showed reversible and concentration‐dependent effects on various electrophysiological parameters. At 3 and 6 mmol/L, but not at 0.1 and 1 mmol/L, 1,8‐cineole significantly diminished the input resistance (Ri) and altered the resting potential (Em) to more positive values. At 6 mmol/L, 1,8‐cineole completely blocked all APs within 2.7 ± 0.6 min (n = 12). In neurons exposed to 3 and 1 mmol/L 1,8‐cineole, the effects regarding excitability varied from complete AP blockade to minor inhibition of AP parameters. The depolarization of Em and the decrease in Ri induced by 6 mmol/L 1,8‐cineole were unaltered by 200 µmol/L niflumic acid, a well known blocker of Ca2+‐activated Cl− currents. 3  Significant correlations (Pearson correlation test) were found between changes in Em and decreases in AP amplitude (r = –0.893; P < 0.00282) and maximum ascendant inclination (r = –0.799; P < 0.0173), but not for maximum descendant inclination (r = 0.598; P < 0.117). Application of current to restore the transmembrane potential equal to control Em values in the presence of 6 mmol/L 1,8‐cineole resulted in the partial recovery of AP. 4  The present study shows that 1,8‐cineole effectively blocks the excitability of SCG neurons, probably through various mechanisms, one of which acts indirectly via depolarization of the neuronal cytoplasmatic membrane.

Effects of 1,8-cineole on Na+ currents of dissociated superior cervical ganglia neurons

1,8-Cineole is a terpenoid present in many essential oil of plants with several pharmacological and biological effects, including antinociceptive, smooth muscle relaxant and ion channel activation. Also, 1,8-cineole blocked action potentials, reducing excitability of peripheral neurons. The objective of this work was to investigate effects of 1,8-cineole on Na(+) currents (INa(+)) in dissociated superior cervical ganglion neurons (SCG). Wistar rats of both sexes were used (10-12 weeks old, 200-300g). SCGs were dissected and neurons were enzymatically treated. To study 1,8-cineole effect on INa(+), the patch-clamp technique in whole-cell mode was employed. 1,8-Cineole (6.0mM) partially blocked INa(+) in SCG neurons. The effect stabilized within ∼150s and there was a partial recovery of INa(+) after washout. Current density was reduced from -105.8 to -83.7pA/pF, corresponding to a decrease to ∼20% of control. 1,8-Cineole also reduced the time-to-peak of INa(+) activation and the amplitude and decay time constants of INa(+) inactivation. Current-voltage plots revealed that 1,8-cineole left-shifted the V1/2 of both activation and inactivation curves by ∼10 and ∼20mV, respectively. In conclusion, we demonstrate that 1,8-cineole directly affects Na(+) channels of the SCG by modifying several gating parameters that are likely to be the major cause of excitability blockade.

Geraniol Induces Antinociceptive Effect in Mice Evaluated in Behavioural and Electrophysiological Models.

Geraniol (GER) is a monoterpene alcohol with various biochemical and pharmacological properties present in the essential oil of more than 160 species of herbs (especially the Cymbopogon genus). In this study, we evaluated the antinociceptive activity of GER in behavioural and electrophysiological in vitro experimental models of nociception using male Swiss mice. GER (12.5, 25 or 50 mg/kg i.p. and 50 or 200 mg/kg p.o.) reduced the number of writhes induced by acetic acid. The opioid antagonist naloxone (5 mg/kg s.c.) administered in mice subsequently treated with GER (25 mg/kg i.p.) did not reverse such antinociceptive activity, suggesting a non‐opioid pathway for the mechanism of action. GER (12.5, 25 and 50 mg/kg i.p.) reduced paw licking time in the second phase of the formalin test. Also, in the glutamate test, GER when administered 50 mg/kg i.p. reduced paw licking time, probably modulating glutamatergic neurotransmission. GER blocked reversibly components of the compound action potential (CAP) recorded in isolated sciatic nerve in a concentration‐ and drug exposure time‐dependent manner: 1 mM to 120 min. for the first component and 0.6 mM to 90 min. for the second component. The IC50 was calculated for the peak‐to‐peak amplitude (PPA) at 0.48 ± 0.04 mM. The conduction velocity was also reduced by exposure to GER starting from the concentration of 0.3 mM for both components of the CAP. In conclusion, it is suggested that GER has antinociceptive activity, especially in pain related to inflammation, and in part related to reduced peripheral nerve excitability.

Essential oil of Croton zehntneri and its main constituent anethole block excitability of rat peripheral nerve.

Croton zehntneri is an aromatic plant native to Northeast Brazil and employed by local people to treat various diseases. The leaves of this plant have a rich content of essential oil. The essential oil of C. zehntneri samples, with anethole as the major constituent and anethole itself, have been reported to have several pharmacological activities such as antispasmodic, cardiovascular, and gastroprotective effects and inducing the blockade of neuromuscular transmission and antinociception. Since several works have demonstrated that essential oils and their constituents block cell excitability and in view of the multiple effects of C. zehntneri essential oil and anethole on biological tissues, we undertook this investigation aiming to characterize and compare the effects of this essential oil and its major constituent on nerve excitability. Sciatic nerves of Wistar rats were used. They were mounted in a moist chamber, and evoked compound action potentials were recorded. Nerves were exposed in vitro to the essential oil of C. zehntneri and anethole (0.1-1 mg/mL) up to 180 min, and alterations in excitability (rheobase and chronaxie) and conductibility (peak-to-peak amplitude and conduction velocity) parameters of the compound action potentials were evaluated. The essential oil of C. zehntneri and anethole blocked, in a concentration-dependent manner with similar pharmacological potencies (IC50: 0.32 ± 0.07 and 0.22 ± 0.11 mg/mL, respectively), rat sciatic nerve compound action potentials. Strength-duration curves for both agents were shifted upward and to the right compared to the control curve, and the rheobase and chronaxie were increased following essential oil and anethole exposure. The time courses of the essential oil of C. zehntneri and anethole effects on peak-to-peak amplitude of compound action potentials followed an exponential decay and reached a steady state. The essential oil of C. zehntneri and anethole caused a similar reduction in conduction velocities of the compound action potential waves investigated. In conclusion, we demonstrated here that the essential oil of C. zehntneri blocks neuronal excitability and that this effect, which can be predominantly attributable to its major constituent, anethole, is important since these agents have several pharmacological effects likely related to the alteration of excitability. This finding is relevant due to the use of essential oils in aromatherapy and the low acute toxicity of this agent, which exhibits other effects of potential therapeutic usefulness.

Essential Oil of Ocimum basilicum L. and (−)-Linalool Blocks the Excitability of Rat Sciatic Nerve

The racemate linalool and its levogyrus enantiomer [(−)-LIN] are present in many essential oils and possess several pharmacological activities, such as antinociceptive and anti-inflammatory. In this work, the effects of essential oil obtained from the cultivation of the Ocimum basilicum L. (EOOb) derived from Germplasm Bank rich in (−)-LIN content in the excitability of peripheral nervous system were studied. We used rat sciatic nerve to investigate the EOOb and (−)-LIN effects on neuron excitability and the extracellular recording technique was used to register the compound action potential (CAP). EOOb and (−)-LIN blocked the CAP in a concentration-dependent way and these effects were reversible after washout. EOOb blocked positive amplitude of 1st and 2nd CAP components with IC50 of 0.38 ± 0.2 and 0.17 ± 0.0 mg/mL, respectively. For (−)-LIN, these values were 0.23 ± 0.0 and 0.13 ± 0.0 mg/mL. Both components reduced the conduction velocity of CAP and the 2nd component seems to be more affected than the 1st component. In conclusion EOOb and (−)-LIN inhibited the excitability of peripheral nervous system in a similar way and potency, revealing that the effects of EOOb on excitability are due to the presence of (−)-LIN in the essential oil.

n5-STZ Diabetic Model Develops Alterations in Sciatic Nerve and Dorsal Root Ganglia Neurons of Wistar Rats

One experimental model of diabetes mellitus (DM) similar to type II DM, called n5-STZ, is obtained by a single injection (via i.p.) of streptozotocin (STZ) in the 5th day of life of newborn rats. The present investigation aimed to characterize alterations in excitability of rat peripheral neurons in n5-STZ model. n5-STZ DM was induced, and electrophysiological evaluation was done at 12th week of rat life. Rats developed glucose intolerance, sensory alteration, and hyperglycemia or near-normoglycemia (21.2 ± 1.6 and 7.4 ± 0.4 mmol/L). In near-normoglycemia group the significant electrophysiological alteration observed was decreased in amplitude of 2nd wave (2nd component, conduction velocity: 48.8 m/s) of compound action potential (CAP) of sciatic nerve. For hyperglycemic rats, decreased excitability, amplitude, and conduction velocity of 2nd CAP component of sciatic nerve were found; a depolarization of resting potential (4-5 mV) and reduction in maximum ascendant and descendant inclinations of action potential were found in DRG neurons but no alteration on Na+ current (INa+). Thus, n5-STZ rats develop alterations in excitability which were related to glycemic levels but were not likely attributable to changes on INa+. Our data confirm that n5-STZ model is a useful model to study type II DM.

1,8-Cineole blocks voltage-gated L-type calcium channels in tracheal smooth muscle

Abstract1,8-Cineole is a cyclic monoterpenoid used in folk medicine for treatment of numerous respiratory diseases and other infections. 1,8-Cineole has anti-inflammatory, antioxidant, and myorelaxant effects, as well as low toxicity. In the present study, the effects of 1,8-cineole on contractility and voltage-gated calcium channels (VGCC) in tracheal smooth muscle were investigated. Intact and dissociated tracheal smooth muscle were used for muscle contraction and patch-clamp recordings, respectively. In experiments involving muscle contraction, 1,8-cineole potentiated contractions at low concentrations and relaxed contractions induced by isotonic K+ at high concentrations. AMTB (a TRPM8 channel blocker) reduced the potentiation induced by 1,8-cineole while indomethacin (a COX inhibitor) did not block this effect. In dissociated myocytes, 1,8-cineole partially blocked Ba2+ currents through VGCC in a concentration-dependent manner. 1,8-Cineole shifted the steady-state activation and inactivation curves to the left and also reduced the current decay time constant. In conclusion, 1,8-cineole has a dual effect on tracheal smooth muscle contraction resulting in a biphasic effect. Our data suggest that the potentiation effect is mediated by activation of TRPM8 channels and the relaxation effect is mediated by the blockage of L-type VGCC.

Hydroxyl Group and Vasorelaxant Effects of Perillyl Alcohol, Carveol, Limonene on Aorta Smooth Muscle of Rats

The present study used isometric tension recording to investigate the vasorelaxant effect of limonene (LM), carveol (CV), and perillyl alcohol (POH) on contractility parameters of the rat aorta, focusing in particular on the structure-activity relationship. LM, CV, and POH showed a reversible inhibitory effect on the contraction induced by electromechanical and pharmacomechanical coupling. In the case of LM, but not CV and POH, this effect was influenced by preservation of the endothelium. POH and CV but not LM exhibited greater pharmacological potency on BayK-8644-induced contraction and on electromechanical coupling than on pharmacomechanical coupling. In endothelium-denuded preparations, the order of pharmacological potency on electrochemical coupling was LM < CV < POH. These compounds inhibited also, with grossly similar pharmacological potency, the contraction induced by phorbol ester dibutyrate. The present results suggest that LM, CV and POH induced relaxant effect on vascular smooth muscle by means of different mechanisms likely to include inhibition of PKC and IP3 pathway. For CV and POH, hydroxylated compounds, it was in electromechanical coupling that the greater pharmacological potency was observed, thus suggesting a relative specificity for a mechanism likely to be important in electromechanical coupling, for example, blockade of voltage-dependent calcium channel.

Melatonin decreases neuronal excitability in a sub-population of dorsal root ganglion neurons

Melatonin, a powerful antioxidant, participates in the regulation of important physiological and pathological processes. We investigated the actions of melatonin on neuronal excitability of intact dorsal root ganglions (DRG) from rats using intracellular recording techniques in current clamps. Melatonin blocked the generation of action potentials in a concentration-dependent manner. Bath applied melatonin (1.0-1000.0 nM) hyperpolarized the resting membrane potential, and increased the input resistance and rheobase. Melatonin also altered the active electrophysiological properties of the action potential, amplitude and maximum descendant inclination, in a statistically significant way. In order to provide evidence on the mechanism of action of melatonin in the DRG, quantitative PCR (qPCR) was performed. Analyses were performed for melatonin membrane receptors, MT1 and MT2, and it was observed that the DRG expresses MT1 receptors. In addition, we noted that the melatonin-induced effects were blocked in the presence of luzindole, a melatonin receptor antagonist. The minimal effective concentrations of melatonin (10.0 nM) and the blockade of effects caused by luzindole suggest that the effects of melatonin are hormonal, and are induced when it binds to MT1 receptors.