Kavalactones and Dihydrokavain Modulate GABAergic Activity in a Rat Gastric-Brainstem Preparation

• Abstract
• Introduction
• Materials and Methods
• Animal preparation
• Plant extract and drugs
• Statistical analysis
• Results
• GABA_A effects on NTS unitary activity
• Kavalactones and dihydrokavain effects on NTS
  unitary activity
• Interaction of kavalactones or dihydrokavain and saclofen or naloxone on NTS unitary activity
• Interaction of kavalactones or dihydrokavain and muscimol on NTS unitary activity
• Discussion
• Acknowledgements
• References

Abstract

Using an in vitro neonatal rat gastric-brainstem preparation, the activity of majority neurons recorded in the nucleus tractus solitarius (NTS) of the brainstem were significantly inhibited by GABA_A receptor agonist, muscimol (30 μM), and this inhibition was reversed by selective GABA_A receptor antagonist, bicuculline (10 μM). Application of kavalactones (300 μg/ml) and dihydrokavain (300 μM) into the brainstem compartment of the preparation also significantly reduced the discharge rate of these NTS neurons (39 % and 32 %, respectively, compared to the control level), and this reduction was partially reversed by bicuculline (10 μM). Kavalactones or dihydrokavain induced inhibitory effects were not reduced after co-application of saclofen (10 μM; a selective GABA_B receptor antagonist) or naloxone (100 nM; an opioid receptor antagonist). Pretreatment with kavalactones (300 μg/ml) or dihydrokavain (300 μM) significantly decreased the NTS inhibitory effects induced by muscimol (30 μM), approximately from 51 % to 36 %. Our results demonstrated modulation of brainstem GABAergic mechanism by kavalactones and dihydrokavain, and suggested that these compounds may play an important role in regulation of GABAergic neurotransmission.

Introduction

Kava (Piper methysticum Forster f.) is a large perennial shrub cultivated in the Southern Pacific islands. Therapeutically, the rhizome of P. methysticum is used to treat anxiety, stress, restlessness and other central nervous system symptoms [1], [2]. Studies in animals show that P. methysticum extracts induce sleep and muscle relaxation [3]. Because of its psychomotor effects, P. methysticum is one of the first botanicals expected to interact with anesthetics [4]. Sedative, anticonvulsive, antispasmodic and central muscular relaxant effects of P. methysticum are attributed to a group of resinous compounds known as kavalactones [2], [5]. While the underlying mechanism is not entirely clear, it appears that kavalactones act on gamma-aminobutyric acid (GABA) and benzodiazepine binding sites in the brain [6].

GABA is an important inhibitory neurotransmitter that mediates the mammalian central nervous system [7]. When GABA is released from the presynaptic site, it can bind to receptors or be taken up by cells and be metabolized. Two major subtypes of GABA receptors, GABA_A and GABA_B receptors, are well investigated [8]. Nucleus tractus solitarius (NTS), the primary relay for visceroceptive information located in the caudal brainstem, is abundant in neuroactive substances, including GABA [9]. It has been shown that GABA receptors possess an inhibitory influence on NTS activity involved in the regulation of cardiovascular and respiratory functions [10], [11]. We previously observed that GABAergic transmission in the brainstem plays an important role in activity modulation of the NTS neurons receiving gastric vagal inputs in an in vitro neonatal rat gastric-brainstem preparation [12]. In the present study, this preparation was used to evaluate the effects of kavalactones and one of its active constituents, dihydrokavain (Fig. [1]), on brainstem GABAergic activities.

Materials and Methods

Animal preparation

The study protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Chicago. Experiments were performed on 2 - 5 days old Sprague-Dawley neonatal rats. After the animal was deeply anesthetized with halothane, a craniotomy was performed and the forebrain was ablated at the caudal border of the pons by transection. The caudal brainstem and cervical spinal cord were isolated by dissection in modified Krebs solution that contained (in mM): NaCl 128.0, KCl 3.0, NaH₂PO₄ 0.5, CaCl₂1.5, MgSO₄ 1.0, NaHCO₃ 21, mannitol 1.0, glucose 30.0 and 4-(2-hydroxyethyl)-1-piperazineethane-sulfonic acid (HEPES) 10.0. The stomach, connected to the esophagus, with the vagus nerves linking it to the brainstem, remained intact and all the other internal organs were removed. The preparation was then pinned down with the dorsal surface facing up on a layer of Sylgard resin (Dow Corning) in a recording chamber. The preparation was continuously bathed and superfused with Krebs solution at 23 °C ± 1 °C. The bathing solution was equilibrated with 95 % O₂ and 5 % CO₂ and adjusted to pH 7.35-7.45 [12], [13].

The gastric vagal branch was dissected out, and a suction microelectrode was placed on the gastric vagal branch from the subdiaphragmatic vagi for electrical stimulation. The nerve fibers were stimulated with single or paired pulses of 200 μA for 0.2 ms at a frequency of 0.5 Hz by a Grass stimulator (model S8800) coupled to a stimulus isolation unit (SIU 7A, Grass Instruments). Single tonic unitary discharges were recorded extracellularly in the NTS by glass microelectrodes filled with 3 M NaCl, which had an impedance of 5 - 15 MΩ. Two types of NTS units were recorded. I. Units responding to the gastric vagal branch stimulation; a collision test was applied to identify orthodromic responses of this type of cells. II. Units not responding to the electrical stimulation of the gastric vagal branches.

In some experiments, for histological identification purposes, the glass microelectrode was filled with 2 % pontamine sky blue in 0.5 M sodium acetate solution. After each unitary recording, current was applied at 5 μA in 10 s on/10 s off cycles for approximately five minutes, with the negative lead connected to the microelectrode.

To investigate solely the central brainstem effects of the test compounds on NTS neurons, a partition was made at the mid-thoracic level of the preparation. An agar seal separated the recording bath chamber into a brainstem compartment and a gastric compartment. Thus, drug application into the brainstem compartment could not have any peripheral actions. During experiments, test compounds were applied to the brainstem compartment for five minutes prior to pharmacological observation to provide sufficient time to reach a steady-state level. Effects of the test compounds on NTS neuronal activity was then evaluated. The NTS neuronal activity observed during pre-trial (control) were compared to post-trial (washout), to confirm that NTS unitary activity returned to the control level after washout. Tachyphylaxis was not evident in our experimental conditions since response to the reapplication of a given concentration of the test compounds 10 min after washout varied by less than five percent.

During each experiment the NTS unitary discharges were amplified with high gain AC-coupled amplifiers (Axoprobe-1A, Axon Instruments), displayed on a Hitachi digital storage oscilloscope (model VC-6525, Hitachi Denshi, Ltd) and recorded on a Vetter PCM tape recorder (model 200, A.R. Vetter Co.).

Plant extract and drugs

Kavalactone extract (from P. methysticum root) was obtained from Alden Botanica LLC (Seattle, WA). The extract was analyzed by high performance liquid chromatography in order to determine the contents of the six major kavalactones. Individual kavalactones in the extract were desmethoxyyangonin (5.8 %), dihydrokavain (22.6 %), dihydromethysticin (11.0 %), kavain (18.4 %), methysticin (15.4 %), and yangonin (6.6 %). Total kavalactones detected was approximately 80 %. Dihydrokavain (> 97 %), a racemic mixture, was obtained from ChromaDex, Inc. (Santa Ana, CA). Muscimol, bicuculline and saclofen were obtained from Research Biochemicals International (Natick, MA). Naloxone was obtained from Sigma (St. Louis, MO).

Statistical analysis

The data from the NTS unitary activity was analyzed on the basis of action potential discharge rate change after the application of the test compounds (trial). The number of action potentials in a given duration were measured under pre-trial, trial and post-trial conditions. The control data (pre-trial) was normalized to 100 %, and NTS neuronal activity during and after trials were compared to the control data. Results were analyzed using Student’s t-test to determine if changes between the two conditions were significantly different. Probability levels less than 0.05 were taken to indicate significant differences.

Results

GABA_A effects on NTS unitary activity

Thirty-nine tonic units receiving gastric vagal input were recorded in the NTS. When GABA_A receptor agonist, muscimol, was applied to the brainstem compartment, the firing rate in the majority of the NTS neurons decreased in a concentration-related fashion. In 30 out of 39 units observed, muscimol (30 μM) produced an inhibitory effect of 53.4 ± 5.2 % (mean ± S.E.) of the control level of the NTS neuronal activity (P < 0.01). Bicuculline (10 μM), a selective GABA_A receptor antagonist, reversed the inhibitory effects of muscimol. The remaining nine NTS cells showed no response to muscimol. The effects of muscimol were also evaluated in another 29 NTS units which did not receive gastric vagal inputs. In 23 out of 29 units observed, muscimol (30 μM) induced 49.2 ± 6.1 % inhibition of the NTS unitary activity (P < 0.01). The inhibitory effects were also antagonized by bicuculline (10 μM) application. The remaining six units showed no response to muscimol. None of the NTS neurons recorded showed activation response to muscimol. Table [1] summarized some of these results.

Kavalactones and dihydrokavain effects on NTS
unitary activity

Effects of kavalactones and dihydrokavain were evaluated in 30 muscimol-sensitive NTS units that received gastric vagal inputs. Kavalactones (300 μg/ml; IC₅₀ = 79 μg/ml) application induced inhibitory effect of 39.1 ± 5.0 % (Fig. [2]). This inhibitory effect was also seen after dihydrokavain (300 μM; IC₅₀ = 93 μg/ml) application (31.5 ± 3.9 %; Fig. [3]). There were significant discharge rate differences between the control recordings and the recordings after kavalactones (300 μg/ml) or dihydrokavain (300 μM) applications (both P < 0.01). Bicuculline (10 μM) partially antagonized kavalactones or dihydrokavain induced inhibitory effects (Figs. [2] and [3]). In addition, similar effects of kavalactones or dihydrokavain application were evaluated in 23 muscimol-sensitive NTS units that did not receive gastric vagal inputs. Kavalactones (300 μg/ml) application induced inhibitory effect of 40.7 ± 5.9 %. This inhibitory effect was also be seen after dihydrokavain (300 μM) application (32.6 ± 4.6 %). There were significant discharge rate differences between the control recording and the recording after kavalactones (300 μg/ml) or dihydrokavain (300 μM) application (both P < 0.01). Bicuculline (10 μM) also partially antagonized these inhibitory effects by kavalactones or dihydrokavain. Muscimol-insensitive NTS units did not show significant response to kavalactones or dihydrokavain.

Interaction of kavalactones or dihydrokavain and saclofen or naloxone on NTS unitary activity

We tested 25 NTS units receiving gastric vagal inputs with inhibitory responses to both kavalactones or dihydrokavain. As shown in Fig. [4], kavalactones-induced inhibitory responses were not significantly reduced after co-application of saclofen (10 μM; a selective GABA_B receptor antagonist) or naloxone (100 nM, a commonly used opioid receptor antagonist). Similar results were obtained in dihydrokavain-induced inhibitory responses.

Interaction of kavalactones or dihydrokavain and muscimol on NTS unitary activity

In this part of the experiment, muscimol effects were evaluated with pretreatment of kavalactones or dihydrokavain. Pretreatment with kavalactones (300 μg/ml) decreased the NTS inhibitory effects induced by muscimol (30 μM) from 51.0 ± 5.9 % to 37.7 ± 3.8 %. Similar results were obtained with dihydrokavain, in which pretreatment with the compound (300 μM) decreased the NTS inhibitory effects induced by muscimol (30 μM) from 51.9 ± 5.3 % to 35.1 ± 4.5 % (Fig. [5]). There were significant discharge rate differences between the muscimol alone and the recording after pretreatment of kavalactones or dihydrokavain (both P < 0.05).

Discussion

Data from clinical studies suggested that P. methysticum extract is superior to placebo as a symptomatic treatment for anxiety [2]. Chemical analysis of P. methysticum extract has identified up to 15 kavalactones [14]. Among them, dihydrokavain possesses a significant anxiolytic effect [15]. Kavalactones may act as a sedative and hypnotic by potentiating GABA inhibitory neurotransmission [16], [17]. It has also been shown that GABAergic transmission has a general depressant action on the central nervous system [18].

Results from previous studies demonstrated that GABA system has an inhibitory influence on NTS activity involved in the baroreceptor inputs and chemoreceptor reflex in different animal species [10]. GABA is also a neurontransmitter in the brainstem, regulating respiratory function [11]. In this study, we observed the inhibitory effects of GABA_A receptor agonist on tonic discharge rates of two types of NTS neurons (i. e., cells receiving or not receiving gastric vagal inputs). We also showed that the tonic activity of the majority of these two types of NTS units was inhibited by kavalactones or dihydrokavain. However, this inhibition was only partially reversed by bicuculline.

Data from this study demonstrated that saclofen, a selective GABA_B receptor antagonist, did not antagonize kavalactones’ inhibitory effect on brainstem neurons. This result is consistent with a previous in vitro study in which no kavalactones’ GABA_B binding sites were observed in the rat neuronal membrane [6]. Although at high concentration, kavalactones were able to bind to brain μ and δ opioid receptors [17], our study did not show that naloxone significantly reversed the inhibitory effects of kavalactones or dihydrokavain at the concentrations used in this study.

In this study, we observed that pretreatment with kavalactones or dihydrokavain significantly decreased the NTS inhibitory effects induced by GABA_A agonist, indicating interactions of kavalactones or dihydrokavain with ligand-bindings of GABA_A receptors. This indicates that the actions of kavalactones may be mediated via the GABAergic system. This inference is supported by a recent binding study, in which Dinh et al. showed interactions of various kavalactones with brain GABA_A receptors [17]. It appears that the regulation of GABAergic neurotransmission is important in the action of kavalactones, and dihydrokavain may be responsible for kavalactones’ central nervous system activity.

P. methysticum or kava is one of the top-selling herbs in the U.S. [2]. In spite of safety concerns of recent reports of liver toxicity related to use of kava products in humans [19], [20], the U.S. Food and Drug Administration has not taken any action against kava products. Data from this study suggest that the modulation of GABAergic mechanisms play a role in the pharmacological effects of kavalactones and dihydrokavain. In addition, since kavalactones interfere with brain GABAergic neurotransmission, presurgical kava use may cause kava-anesthetic interaction in surgical patients [4].

Acknowledgements

The authors wish to thank Ms. Spring Maleckar for her technical assistance. This work is support in part by the Tang Family Foundation, Brain Research Foundation, and NIH grants AT00381 and AT00563.

References

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Chun-Su Yuan, MD PhD

Department of Anesthesia & Critical Care

The Pritzker School of Medicine

University of Chicago

5841 S. Maryland Avenue, MC 4028

Chicago, Illinois 60637

U.S.A.

Phone: +1-773-702-1916

Fax: +1-773-834-0601

Email: cyuan@midway.uchicago.edu

References

• 1 Volz H P, Kieser M. Kava-kava extract WS 1490 versus placebo in anxiety disorders - a randomized placebo-controlled 25-week outpatient trial.  Pharmacopsychiatry. 1997;  30 1-5
  PubMedSearch in Google Scholar
• 2 Pittler M H, Ernst E. Efficacy of kava extract for treating anxiety: Systematic review and meta-analysis.  Journal of Clinical Psychopharmacology. 2000;  20 84-9
  CrossrefPubMedSearch in Google Scholar
• 3 Robbers J E, Tyler V E. Nervous system disorders.  In: Tyler’s Herbs of Choice. New York; Haworth Press, Inc 1999: 154-7
  Search in Google Scholar
• 4 Ang-L. M K, Moss J, Yuan C S. Herbal medicines and perioperative care.  JAMA. 2001;  286 208-16
  CrossrefPubMedSearch in Google Scholar
• 5 Singh Y N. Kava: An overview.  Journal of HerbalGram. 1997;  39 33-55
  PubMedSearch in Google Scholar
• 6 Davies L P, Drew C A, Duffield P, Johnston G A, Jamieson D D. Kava pyrones and resin: Studies on GABA_A, GABA_B and benzodiazepine binding sites in rodent brain.  Pharmacology & Toxicology. 1992;  71 120-6
  PubMedSearch in Google Scholar
• 7 Bormann J, Feigenspan A. GABA_C receptors.  Trends in Neurosciences. 1995;  18 515-9
  CrossrefPubMedSearch in Google Scholar
• 8 Johnston G A. GABA_C receptors: Relatively simple transmitter-gated ion channels?.  Trends in Pharmacological Sciences. 1996;  17 319-23
  CrossrefPubMedSearch in Google Scholar
• 9 Maley B E. Immunohistochemical localization of neuropeptides and neurotransmitters in the nucleus solitarius.  Chemical Senses. 1996;  21 367-76
  PubMedSearch in Google Scholar
• 10 Ruggeri P, Cogo C E, Picchio V, Molinari C, Ermirio R, Calaresu F R. Influence of GABAergic mechanisms on baroreceptor inputs to nucleus tractus solitarii of rats.  American Journal of Physiology. 1996;  271 931-6
  PubMedSearch in Google Scholar
• 11 Shao X M, Feldman J L. Respiratory rhythm generation and synaptic inhibition of expiratory neurons in pre-Botzinger complex: Differential roles of glycinergic and GABAergic neural transmission.  Journal of Neurophysiology . 1997;  77 1853-60
  PubMedSearch in Google Scholar
• 12 Yuan C S, Liu D, Attele A S. GABAergic effects on nucleus tractus solitarius neurons receiving gastric vagal inputs.  Journal of Pharmacology and Experimental Therapeutics . 1998;  286 736-41
  PubMedSearch in Google Scholar
• 13 Yuan C S, Attele A S, Dey L, Xie J T. Gastric effects of cholecystokinin and its interaction with leptin on brain stem neuronal activity.  Journal of Pharmacology and Experimental Therapeutics. 2000;  295 177-82
  PubMedSearch in Google Scholar
• 14 Smith R M. High-performance liquid chromatography of kava lactones from Piper methysticum .  Journal of Chromatography.. 1984;  283 303-8
  CrossrefPubMedSearch in Google Scholar
• 15 Smith K K, Dharmaratne H RW, Feltenstein M W, Broom S L, Roach J T, Nanayakkara N PD, Khan I A, Sufka K J. Anxiolytic effects of kava extract and kavalactones in the chick social separation-stress paradigm.  Psychopharmacology. 2001;  155 86-90
  CrossrefPubMedSearch in Google Scholar
• 16 Pepping J. Kava: Piper methysticum .  American Journal of Health System and Pharmarcy. 1999;  56 957-60
  PubMedSearch in Google Scholar
• 17 Dinh L D, Simmen U, Bueter K B, Bueter B, Lundstrom K, Schaffner W. Interaction of various Piper methysticum cultivars with CNS receptors in vitro .  Planta Medica. 2001;  67 306-11
  Thieme ConnectPubMedSearch in Google Scholar
• 18 Cooper J R, Blorr F E, Roth R H. The Biochemical Basis of Neuropharmacology. Oxford University Press New York; 1986
  Search in Google Scholar
• 19 Russmann S, Lauterburg B H, Helbling A. Kava hepatotoxicity.  Annals of Internal Medicine . 2001;  35 68-9
  PubMedSearch in Google Scholar
• 20 Escher M, Desmeules J, Giostra E, Mentha G. Hepatitis associated with kava, a herbal remedy for anxiety.  British Medical Journal. 2001;  322 139
  PubMedSearch in Google Scholar

Chun-Su Yuan, MD PhD

Department of Anesthesia & Critical Care

The Pritzker School of Medicine

University of Chicago

5841 S. Maryland Avenue, MC 4028

Chicago, Illinois 60637

U.S.A.

Phone: +1-773-702-1916

Fax: +1-773-834-0601

Email: cyuan@midway.uchicago.edu