Introduction

Pain is a disagreeable sensory and emotional experience which begins in conjunction with tissue damage or injury. Hence, pain constitutes a kind of biological defense sys-tem, which is important for the maintenance of life (1).

Opioid analgesics, non-steroidal anti-inflammatory drugs (NSAIDs), or local anesthetics are among the cur-rently used agents for controlling pain. Analgesics were initially developed for acute pain. Local anesthetics have been in clinical use for a long time for this purpose. Levobupivacaine and bupivacaine, two long-acting local anesthetics, have also been used for the treatment

of acute and chronic pain (2). But, the cellular mecha-nisms of the antinociceptive effects of local anesthetics, especially the novel agent levobupivacaine, are not well documented.

Bupivacaine and levobupivacaine are among the most efficacious of the available local anesthetics with long action (3–5 h). But the evident cardiotoxic effects of bupivacaine limit its use. To avoid this limiting side effect, a chemically related compound, levobupivacaine, was developed (3,4).

Dorsal root ganglion (DRG) neurons are primary afferent neurons that transmit peripheral stimuli to the neurons in the dorsal horn of the spinal cord.

Journal of Receptors and Signal TransductionJournal of Receptors and Signal Transduction, 2010; 30(2): 115–120

2010

30

2

115

120

Address for Correspondence: Ahmet Ayar, Department of Physiology, Faculty of Medicine (TIP FAKULTESI), Karadeniz Technical University, TR23119 Trabzon, Turkey. E-mail: aayar61@yahoo.com

06 January 2010

17 January 2010

18 January 2010

1079-9893

1532-4281

© 2010 Informa UK Ltd

10.3109/10799891003630614

r e s e a r c h a r T I c L e

Effects of levobupivacaine and bupivacaine on intracellular calcium signaling in cultured rat dorsal root ganglion neurons

Mete Ozcan1, Ahmet Ayar2, Ergul Alcin3, Sibel Ozcan4, and Selim Kutlu3

1Department of Biophysics, Faculty of Medicine, Firat University, Elazig, Turkey, 2Department of Physiology, Faculty of Medicine, Karadeniz Technical University, Trabzon, Turkey, 3Department of Physiology, Faculty of Medicine, Firat University, Elazig, Turkey, and 4Department of Anaesthesiology, Faculty of Medicine, Firat University, Elazig, Turkey

abstractBupivacaine and levobupivacaine have been shown to be effective in the treatment of pain as local anes-thetics, although the mechanisms mediating their antinociceptive actions are still not well understood. The aim of this study was to investigate the effects of bupivacaine and levobupivacaine on intracellular calcium ([Ca2+]i) signaling in cultured rat dorsal root ganglion (DRG) neurons. DRG neuronal cultures loaded with 5 μM Fura-2/AM and [Ca2+]i transients for stimulation with 30 mM KCl (Hi K+) were assessed by using fluores-cent ratiometry. DRGs were excited at 340 and 380 nm, emission was recorded at 510 nm, and responses were determined from the change in the 340/380 ratio (basal-peak) for individual DRG neurons. Data were analyzed by using Student’s t-test. Levobupivacaine and bupivacaine attenuated the KCl-evoked [Ca2+]i transients in a reversible manner. [Ca2+]i increase evoked by Hi K+ was significantly reduced to 99.9 ± 5.1% (n = 18) and 62.5 ± 4.2% (n = 15, P < 0.05) after the application of 5 and 50 µM levobupivacaine, respectively. Bupivacaine also inhibited Hi K+-induced [Ca2+]i responses, reduced to 98.7 ± 4.8% (n = 10) and 69.5 ± 4.5% (n = 9, P < 0.05) inhibition of fluorescence ratio values of Hi K+-induced responses at 5 and 50 μM, respectively. Our results indicate that bupivacaine and levobupivacaine, with no significant differences between both agents, attenuated KCl-evoked calcium transients in a reversible manner. The inhibition of calcium signals in DRG neurons by levobupivacaine and bupivacaine might contribute to the antinociceptive effects of these local anesthetics.

Keywords: Bupivacaine; calcium imaging; dorsal root ganglia; levobupivacaine; local anesthetic; pain

RST

463570

(Received 06 January 2010; revised 17 January 2010; accepted 18 January 2010)

ISSN 1079-9893 print/ISSN 1532-4281 online © 2010 Informa UK LtdDOI: 10.3109/10799891003630614 http://www.informahealthcare.com/rst

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116 Mete Ozcan et al.

In recent years, the DRG model has been extensively used to investigate cellular mechanism of nociception. In chronic pain conditions, DRG neurons can become hyperexcitable, produce spontaneous action poten-tials, or fire at abnormal high frequency. In spinal and epidural anesthesia, the administration of high concentrations of local anesthetics cause peripheral nerve block which is generated through a process involving the DRG neurons (5).

The mechanism underlying the inhibition of pain transmission in peripheral nerves, including DRG neu-rons, by local anesthetics is generally explained by a blockade of different types of voltage-gated Na+ channels (6). However, local anesthetics effects also involve both calcium and potassium channels (7,8).

Calcium channels play an important role in action potential firing of sensory neurons. The structure of voltage-dependent Ca2+ channels closely resembles that of Na+ channels. In addition, Ca2+ currents in DRG neurons are blocked by the local anesthetics. Yet, current findings indicate that the inhibition of calcium influx by local anesthetics through inhibiting voltage-gated Ca2+ channels may contribute to anesthetic mechanisms. Furthermore, a good correlation has been suggested between analgesic action and Ca2+ channel blockade (7,9,10).

Although the effects of many other local anesthetics on intracellular calcium homeostasis have already been shown (7), the effects of levobupivacaine (an optical bupivacaine isomer) on intracellular calcium homeos-tasis in cultured DRG neurons have not been investigated previously.

Hence, the aim of this study was to investigate the effects of bupivacaine and levobupivacaine on depolari-zation-induced calcium signaling in cultured rat primary sensory neurons.

Materials and methods

The protocol of this study was approved by the Institutional Review Committee for animal research.

Cell culture

Primary cell culture of DRG neurons were obtained from 2-day-old Wistar rats. Rats were decapitated, dorsal root ganglia were removed, and cells were dissociated follow-ing enzymatic and mechanical procedures. Enzymatic treatment involved incubation with 0.125% collagenase (Sigma) for 13 min and 0.25% trypsin for 6 min (Sigma), respectively. DRG neurons were then isolated by mechan-ical trituration and plated on laminin-poly-d- lysine–coated glass coverslips and bathed in Neurobasal-A

medium (Gibco, Paisley, Scotland), supplemented with 2% B27, 1% Glutamax, penicillin (5000 IU/mL), strep-tomycin (5000 μg/mL), and nerve growth factor (NGF; 20 ng/mL; Sigma, Poole, England). The cultures were incubated at 37°C in humidified air containing 5% CO

2,

and all calcium imaging experiments were performed from at least 5–6 h of incubation.

Calcium imaging

The cells were loaded with calcium-sensitive dye Fura-2/AM ester (5 µM; Molecular Probes Inc., 1 mM stock in DMSO) by incubation for 60 min in standard recording medium containing (in mM) the following: NaCl, 130; CaCl

2, 2; KCl, 3; MgCl

2, 0.6; NaHCO

3, 0.6;

HEPES, 10; glucose, 4. The pH and osmolarity were adjusted to 7.4 and 320 mOsm/L with NaOH and sucrose, respectively.

After loading, the cells were washed 3–4 times for 20 min with standard recording medium to remove the extracellu-lar Fura-2/AM and to allow cytoplasmic de-esterification of the Ca2+-sensitive fluorescent dye. The cells were viewed under an inverted Nikon TE 2000 S microscope (S-fluor, 40× oil, 1.3 NA) attached with a CCD camera (ORCA 285; Hamamatsu Photonics, Hamamatsu, Japan). The fluo-rescence ratiometric images were obtained at excitations 340 and 380 nm and at emission 510 nm (Fura-2 Filter Set; Semrock Brightline). Filters were changed by using a com-puter controlled filter wheel (Sutter Instruments, USA) placed between the Xenon (300 Watt; Sutter Instruments) light source and the microscope.

The neurons were stimulated with standard recording medium containing high K+ (30 mM), which produced depolarization, activation of voltage-gated Ca2+ chan-nels, and large transient increases in [Ca2+]

i. Cells were

stimulated three times with consistent duration of Hi K+ application in each experimental run.

Bupivacaine and levobupivacaine were tested on response to the second stimulus by 5 min pretreatment before second application of Hi K+. The actions of bupi-vacaine and levobupivacaine on the peak amplitude of the fluorescence ratio (340/380 nm) were measured. All imaging experiments were performed in the dark at room temperature.

Glass coverslips with Fura-2-loaded DRG neurons were mounted in an imaging/perfusion chamber equipped with perfusion valve system (Warner Instruments, Hamden, CT, USA), which was mounted and viewed through an inverted microscope (Nikon TE2000S, Japan). The bath volume of the chamber was 600 µL.

The Student’s t-test for unequal variance was used to determine significance. All values are presented as the mean ± SEM. For all analyses, P < 0.05 was accepted as evidence of significance.

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Levobupivacaine inhibits calcium signaling sensory in neurons 117

Results

Treatment with levobupivacaine and bupivacaine alone did not evoke any [Ca2+]

i change in DRG neurons. And then

under control conditions three consistent transient increase in [Ca2+]

i were observed in response to depolarization of

the membrane by application of extracellular recording medium containing 30 mM KCl. Levobupivacaine and bupivacaine (5 and 50 µM) were applied during the second Hi K+-evoked depolarization. When Hi K+ was applied for second time following full recovery of intracellular Ca2+ the response was similar in nature and amplitude to the first application (Figures 1–3).

When the effects of low dose of levobupivacaine (5 µM) was tested, fluorescence ratio was reduced to 99.9 ± 5.1% compared with [Ca2+]

i elicited by its first

application (Figure 1A; n = 18). Peak [Ca2+]i response

to Hi K+ was significantly reduced to 62.5 ± 4.2% by application of 50 µM levobupivacaine (Figure 2A; P < 0.05, n = 15).

Bupivacaine also inhibited [Ca2+]i responses, causing

reduction of Hi K+-induced [Ca2+]i transients to 98.7 ± 4.8%

(n = 10) and 69.5 ± 4.51 (n = 9) after the application of 5 and 50 µM bupivacaine, respectively (Figure 3A and 3C).

When the area under the curve (AUC) for the Hi K+-elicited [Ca2+]

i transients was considered, consistent with

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Figure 1. Effects of 5 µM levobupivacaine on 30 mM KCl (Hi K+)-evoked Ca2+ transients in cultured rat DRG neurons. Fluorescence calcium responses are graphed as a percentage of mean Fura-2 fluorescence ratio increase at the (A) peak level and (B) area under the curve (AUC) of Ca2+ transients after stimulation with Hi K+ compared with baseline. The first peak represents response to first application of Hi K+, second transient represents the response to application of Hi K+ after 5 min pretreatment with levobupivacaine, and the last peak represents response to the third application of Hi K+ alone. The bottom inset shows a typical tracing among the protocol from a representative single cell. Similar results were obtained from different experiments and the mean results (expressed as mean ± SEM) are derived from three different dishes.

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118 Mete Ozcan et al.

the mean peak amplitude data, the AUC of [Ca2+]i tran-

sients was significantly reduced to 97.8 ± 3.95% (n = 18) and 71.15 ± 4.57% (n = 15) after the application of 5 µM and 50 µM levobupivacaine (Figure 1B and 2B); and 99.97 ± 5.41% (n = 10) and 75.39 ± 5.6% (n = 9) after the application of 5 and 50 µM bupivacaine, respectively (Figure 3B and 3D).

Discussion

The results of this study demonstrate for the first time in cultured rat sensory neurons the reversible inhibition of intracellular calcium signaling by levobupivacaine and bupivacaine.

The use of DRG neurons in culture provides an eli-gible model for pain and analgesia research which has led to tremendous advances in the understanding of nociceptive transmission and development of analgesic drugs. In the DRG, an intracellular signaling pathway is the premium target in pain research that is thematic to the neuroplastic changes that occur during chronic pain conditions (11) as well as mediating acute nociceptive signaling (12).

Local anesthetics have been used clinically for a long time for anesthesia as well as for the treatment of acute and chronic pain (13). The use of local anesthetics is an important treatment considered for many patients with acute and chronic pain conditions including chronic cancer pain (14).

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Figure 2. An aliquot of 50 µM levobupivacaine inhibits 30 mM KCl (Hi K+)-evoked Ca2+ transients in cultured rat DRG neurons. Fluorescence calcium responses are graphed as a percentage of mean Fura-2 fluorescence ratio increase (A) at the peak level and (B) area under curve (AUC) of Ca2+ transients after stimulation with Hi K+ compared with baseline. The first peak represents response to first application of Hi K+, second transient represents the response to application of Hi K+ after 5 min pretreatment with levobupivacaine and the last peak represents response to the third application of Hi K+ alone. The bottom inset shows a typical tracing among the protocol from a representative single cell. Similar results were obtained from different experiments and the mean results (expressed as mean ± SEM) are derived from three different dishes.

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Levobupivacaine inhibits calcium signaling sensory in neurons 119

The cellular mechanism mediating the antino-ciceptive activity of bupivacaine is not thoroughly documented but several putative mechanisms have been suggested (15–17). Bupivacaine binds and blocks sodium influx into nerve cells and thereby prevents depolarization. It has been shown that blockage of TTX-resistant Na+ currents by bupivacaine reduce firing frequency in DRG neurons (15). Bupivacaine reversibly decreased both Ca2+ channel current magnitude and a specific μ-opioid receptor agonist induced the inhibition of Ca2+ channel currents in sen-sory neurons (18). Furthermore, there are evidence that local anesthetics also inhibit calcium entry in DRG neurons (e.g., tetracaine, bupivacaine, n-butyl-p- aminobenzoate) and dorsal horn neurons (e.g., ropivacaine) (7,19,20). Our bupivacaine results are in agreement with the previous study (7) which clearly indicates that bupivacaine significantly inhibited the depolarization-induced increase in intracellular

free calcium levels in cultured rat DRG neurons. The cellular mechanism(s) of analgesic activity of lev-obupivacaine is also not known (21). Our study shows that levobupivacaine inhibited the intracellular cal-cium signaling in DRG neurons similarly with the effect of bupivacaine. And, this may indicate a possible mechanism of the analgesic effect of levobupivacaine, which have been clinically proven (21).

The inhibitory effects of many agents on calcium entry into DRG neurons may mediate the antinociceptive action. Recently, gabapentin and levetiracetam, new class of anticonvulsant, are introduced for the management of neuropathic pain (22–24). In DRG, influx of Ca2+ via Ca2+ channels modulate the release of neurotransmitters and gabapentin and levetiracetam reduce the calcium entry and thereby elicit analgesic effect and can be useful in the treatment of neuropathic pain (25,26). Indeed, the antiepileptic drug gabapentin has been widely used for treating neuropathic pain (22,27).

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Figure 3. The effects of 5 µM and 50 µM bupivacaine on KCl-evoked Ca2+ transients in cultured rat DRG neurons. Fluorescence calcium responses are graphed as a percentage of mean Fura-2 fluorescence ratio increase at the peak level (A and C) and area under the curve (AUC) of Ca2+ tran-sients (B and D) after stimulation with Hi K+ compared with baseline. The first peak represents response to first application of 30 mM KCl (Hi K+), second transient represents the response to application of Hi K+ after 5 min pretreatment with bupivacaine, and the last peak represents response to the third application of Hi K+ alone. Similar results were obtained from different experiments and the mean results (expressed as mean ± SEM) are derived from two different dishes.

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120 Mete Ozcan et al.

The inhibition of calcium signaling by levobupi-vacaine and bupivacaine may cause the reduction of excitability in DRG and in peripheral sensory nerve fibers. A good correlation between local anesthetic bupivacaine and levobupivacaine potencies to inhibit intracellular calcium transients and their anesthetic potencies further implies that the inhibition of intracel-lular calcium transients may contribute to the analgesic mechanisms.

In conclusion, we found that bupivacaine and lev-obupivacaine, with no significant differences between both agents, reduced the increase in intracellular calcium, induced by membrane depolarization, in a reversible manner. The detailed mechanisms underly-ing the actions of levobupivacaine and bupivacaine remain unclear and further investigations are needed for understanding the cellular mechanisms of these local anesthetics.

Acknowledgements

The authors would like to thank Turkish Scientific Technical Research Organization for providing laboratory equipment (TUBITAK Project No: 104S514).

Declaration of interest

The authors declare no conflict of interest with regard to data presented in this article.

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