Regulatory effects of anandamide on intracellular Ca2+ concentration increase in trigeminal ganglion neurons
Department of Anesthesiology, Tongji Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, China
Jingzhou Central Hospital, Jingzhou, Hubei Province, China
Department of Physiology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, China
Department of Bioengineering, Wuhan Institute of Engineering, Wuhan, Hubei Province, China
Corresponding author: Xuehong Cao, Department of Physiology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China; Department of Bioengineering, Wuhan Institute of Engineering, Wuhan 430023, Hubei Province, China caoxuehong@gmail.com.. Lieju Liu, Department of Bioengineering, Wuhan Institute of Engineering, Wuhan 430023, Hubei Province, China, liejuliu@gmail.com.Abstract
Activation of cannabinoid receptor type 1 on presynaptic neurons is postulated to suppress neurotransmission by decreasing Ca2+ influx through high voltage-gated Ca2+ channels. However, recent studies suggest that cannabinoids which activate cannabinoid receptor type 1 can increase neurotransmitter release by enhancing Ca2+ influx in vitro. The aim of the present study was to investigate the modulation of intracellular Ca2+ concentration by the cannabinoid receptor type 1 agonist anandamide, and its underlying mechanisms. Using whole cell voltage-clamp and calcium imaging in cultured trigeminal ganglion neurons, we found that anandamide directly caused Ca2+ influx in a dose-dependent manner, which then triggered an increase of intracellular Ca2+ concentration. The cyclic adenosine and guanosine monophosphate-dependent protein kinase systems, but not the protein kinase C system, were involved in the increased intracellular Ca2+ concentration by anandamide. This result showed that anandamide increased intracellular Ca2+ concentration and inhibited high voltage-gated Ca2+ channels through different signal transduction pathways.
Introduction
N-arachidonoylethanolamine (anandamide) is referred to as the first ‘endocannabinoid’ described, and anandamide signaling has been reported to be an inhibitor of axon regeneration. Anandamide acts primarily on cannabinoid receptor type 1 (CB1)[1], cannabinoid receptor type 2 (CB2)[2], and ionotropic receptors, including the transient receptor potential (TRP) vanilloid type 1 (TRPV1)[345], TRP ankyrin type 1 (TRPA1) and TRP melastatin type 8 (TRPM8) channels[6]. Anandamide can also directly modulate various other ion channels[578910].
CB1 and CB2 are members of the subfamily of G-protein-coupled receptors and predominantly couple to Gi/o[111] to produce multiple cellular effects, such as the inhibition of adenylate cyclase and of voltage-gated calcium channels[121314], regulation of potassium currents[151617], and increase of Ca2+ influx via Gs[18] and Gq[19]. TRPV1 is a polymodal sensor of noxious stimuli including heat, hydrogen ions and capsaicin[20]. CB1[21] and TRPV1, but not CB2[21] are expressed in presynaptic primary neurons such as those in the trigeminal ganglion. CB1 is localized to presynaptic nerve terminals[22] and contributes to the regulation of neuronal excitability and neurotransmitter release by modulating Ca2+ signals. TRPV1 activation causes Ca2+ to enter into the cell and promotes neurotransmitter release. Studies have demonstrated that anandamide can activate TRPV1 by binding to cytosolic sites[23]. The co-expression and close distribution of CB1 and TRPV1 in primary sensory neurons[24252627] allows cross-talk between these two receptors, which further complicates the role of anandamide in nociception and antinociception. The present study will determine which receptor contributes to the effect of low and high concentrations of anandamide in small trigeminal ganglion neurons.
It is a widely accepted general hypothesis that endocannabinoids inhibit neurotransmitter release from primary afferent neurons by reducing Ca2+ influx via an inhibitory action on high voltage-gated Ca2+ channels. This hypothesis is supported by evidence that CB1 activation by anandamide on primary nociceptive neurons causes antinociception by reducing high voltage-activated Ca2+ channel activity[121314] and Ca2+ influx[28], thus inhibiting neurotransmission. In contrast, some studies suggest that cannabinoids can promote Ca2+ influx and hence increase neurotransmitter release in vitro[1925293031]. How endocannabinoids may cause such opposing effects in intracellular Ca2+ concentration and neuron excitability is unclear. High voltage-gated calcium channels and ligand-gated channels, two important contributors to intracellular Ca2+ concentration, will also be investigated. The present study tests the above hypothesis by determining the effect of anandamide on high voltage-activated Ca2+ currents (IHVA), Ca2+ influx and intracellular Ca2+ concentration, as well as the underlying mechanisms, in small trigeminal ganglion neurons.
Results
Anandamide-evoked inward currents
Whole-cell patch-clamp recordings were carried out in rat trigeminal ganglion neurons with bath perfusion of 0.1, 0.3, 1, 3, 10, 30 and 100 μmol/L anandamide at a range of holding potentials from −60 mV to +60 mV (Figure 1). Reversal potentials were around 0 mV. Anandamide-evoked inward and outward currents at holding potentials of −60 mV and +60 mV, respectively, were dose-dependent (Figure 2).
Inhibition of IHVA by anandamide
Bath application of anandamide (0.001, 0.01, 0.1, 1, 10, 30 μmol/L) inhibited IHVA in a concentration-dependent manner, by 4.62 ± 0.88%, 6.24 ± 2.57%, 18.17 ± 0.99%, 31.30 ± 3.21%, 64.73 ± 1.95% and 55.19 ± 2.06%, respectively (n = 4–11). The effect of anandamide was partially reversed after washout. The Hill equation was applied to the dose-response curve and revealed that the half-maximal inhibitory concentration (IC50) of anandamide was 0.92 μmol/L. To confirm whether other CB1 agonists mimicked the inhibition of anandamide, we tested the effect of WIN 55,212-2 (10 μmol/L) on IHVA. The inhibition of IHVA by 10 μmol/L WIN 55,212-2 was 55.78 ± 6.07%. Anandamide at 1 μmol/L reduced current amplitudes (Figure 3A, B) but did not cause a significant shift of the activation curve (Figure 3C). However, a hyperpolarization shift of almost 14 mV (n = 8; P < 0.05) was observed in the h-infinity curve (Figure 3E).
Roles of cannabinoid and vanilloid receptors in the inhibition of IHVA by anandamide
Since anandamide activates TRPV1, CB1 and CB2 receptors, we tested whether capsazepine, AM251 and AM630, selective antagonists at the three receptors, respectively, could reverse the effect of anandamide on IHVA. Capsazepine (10 μmol/L), AM251 (10 μmol/L), AM630 (10 μmol/L), or anandamide (1 and 10 μmol/L) were added to the bath solution during whole-cell patch clamp recordings in rat trigeminal ganglion neurons. First, we examined how pre-incubation of the cells for 3 minutes with capsazepine, a competitive antagonist at TRPV1, would affect the inhibition induced by anandamide (Figure 4A, D). Pre-incubation with capsazepine did not affect the inhibition of IHVA by 1 μmol/L anandamide (n = 10, P > 0.05), but abolished the effect of 10 μmol/L anandamide (n = 12, P < 0.05). These data indicate that TRPV1 activation is involved in the inhibition of IHVA by high-concentration anandamide. AM251 was co-applied with anandamide (1 or 10 μmol/L) (Figure 4B, E). Low-dose (n = 7, P < 0.05) and high-dose (n = 4, P < 0.05) anandamide-induced inhibition was reversed by AM251 (Figure 4G), indicating that activation of CB1 is essential for the negative modulation of IHVA by anandamide in rat trigeminal ganglion neurons.
Pre-incubation for 3 minutes with AM630 did not affect IHVA inhibition induced by 1 μmol/L (Figure 4C, G; P > 0.05) or 10 μmol/L (Figure 4F, G; P > 0.05) anandamide, indicating a lack of CB2 receptors in rat trigeminal ganglion neurons[21].
Inhibition of IHVA by capsaicin
As described above, inhibition of IHVA by high-concentration anandamide was reversed by the TRPV1 antagonist capsazepine, suggesting that TRPV1 is involved in this effect. We further tested whether IHVA could be directly inhibited by the TRPV1 agonist, capsaicin. Capsaicin and capsazepine were applied by bath perfusion. IHVA was reversibly inhibited by capsaicin in a dose-dependent manner (Figure 5). Similar to the effect observed with high-concentration anandamide, the decrease in IHVA by capsaicin was reversed by 10 μmol/L capsazepine. In addition, IHVA was not inhibited by 10 μmol/L capsazepine (Figure 6).
Characterization of signal transduction pathways mediating the inhibition of IHVA via cannabinoid type-1 receptor activation by low-concentration anandamide
Three important signal transduction systems were examined using specific agonists and antagonists to test whether these systems participated in the inhibition of IHVA by CB1 receptors activated by 1 μmol/L anandamide.
Whole-cell patch clamp measurements in trigeminal ganglion neurons revealed that application of 1 μmol/L KT5720, an inhibitor of cyclic adenosine monophosphate -dependent protein kinase A, significantly attenuated the inhibition of IHVA by 1 μmol/L anandamide (Figure 7). Incubation with an antagonist of protein kinase C, bisindolylmaleimide (1 μmol/L for 10 minutes) and an inhibitor of cyclic guanosine monophosphate (cGMP)-dependent protein kinase, Rp-8-Br-cGMP (1 μmol/L for 10 minutes) also significantly attenuated anandamide-induced inhibition of IHVA.
Anandamide increased intracellular Ca2+ concentration via a direct influx of extracellular calcium into rat trigeminal ganglion neurons
Calcium imaging was used to examine the effect of anandamide on intracellular Ca2+ concentration in the presence of solutions containing either 0 or 2 mmol/L Ca2+ (Figure 8A). In the absence of extracellular calcium (buffered by 10 mmol/L ethylene glycol bis(alpha-aminoethyl ether)-N,N′-tetraacetic acid to decrease extracellular Ca2+ concentrations), the rise in intracellular Ca2+ concentration induced by anandamide was notably decreased (Figure 8B; anandamide([Ca2+]+): 20.33 ± 2.10%, n = 19; anandamide([Ca2+]−): 4.22 ± 1.06%, n = 48; P < 0.01). Thus, the anandamide-induced rise in intracellular Ca2+ concentration is dependent on Ca2+ influx from the extracellular space.
Signaling pathways mediating the increase in intracellular Ca2+ concentration after cannabinoid receptor activation by low-concentration anandamide
Calcium imaging was used to evaluate the possible contribution of cAMP-dependent protein kinase, protein kinase C, and cGMP-dependent protein kinase systems to the observed effect on intracellular Ca2+ concentration after CB1 activation. The effects of 1 μmol/L anandamide on intracellular Ca2+ concentrations in trigeminal ganglion neurons pre-treated with KT5720 (an inhibitor of cAMP-dependent protein kinase), staurosporine (an inhibitor of protein kinase C) and KT5823 (an inhibitor of cGMP-dependent protein kinase) were investigated. Figure 8D showed that staurosporine (10 μmol/L for 15 minutes) had no effect on the rise in intracellular Ca2+ concentration after anandamide application (n = 13, P > 0.05). Pretreatment with KT5720 (10 μmol/L for 10 minutes) and KT5823 (10 μmol/L for 10 minutes) markedly attenuated the response of 1 μmol/L anandamide Figure 8D. These data suggest that the cAMP-dependent protein kinase and cGMP-dependent protein kinase systems, but not the protein kinase C system, are involved in the increase in intracellular Ca2+ concentration with 1 μmol/L anandamide.
Discussion
It is well established that presynaptic CB1 receptor activation has an inhibitory role on high voltage-activated Ca2+ channels, resulting in a decrease in Ca2+ influx and an inhibition of neurotransmitter release. In contrast, results from the present study show that anandamide causes a dose-dependent elevation of intracellular Ca2+ concentration, triggered by anandamide-evoked currents, but not modulation of high voltage-activated Ca2+ channels by anandamide. Furthermore, anandamide-induced inhibition of the high voltage-activated Ca2+ channels and the increase in the intracellular Ca2+ concentration are demonstrated to be two separate events differing in signal transduction pathways but having similar effects via the same receptors. Thus, our data suggest that anandamide, as an endocannabinoid, might exert an excitatory effect by increasing intracellular Ca2+ concentration, triggered by anandamide-evoked currents.
It has been well documented that the endocannabinoid anandamide, and its synthetic analogue methanandamide, have dual effects. At low concentrations, anandamide selectively activates CB1 receptors, while at high concentrations it activates excitatory TRPV1 receptors[24]. In pre-contracted strips of rat hepatic artery, rat small mesenteric artery and guinea-pig basilar artery, vasorelaxation induced by high-concentration anandamide is antagonized by the TRPV1 antagonist capsazepine, but not by the CB1 receptor antagonist SR141716 (0.3 μmol/L)[4]. Further evidence indicates that anandamide not only acts on transfected TRPV1 receptors to produce membrane currents and increase the intracellular Ca2+ concentration[432], but also acts on naturally-expressed TRPV1 receptors in neonatal rat dorsal root ganglia to produce membrane currents[33]. Here, we present strong evidence that anandamide selectively activates the CB1 receptor at low concentrations in small trigeminal ganglion neurons. At high concentrations, anandamide activates both CB1 and TRPV1 receptors, inhibiting IHVA. To confirm that high-concentration anandamide inhibited IHVA via TRPV1 receptor activation, we compared the effects of high-concentration anandamide and the TRPV1 agonist capsaicin. We found that the inhibition induced by high-concentration anandamide and by capsaicin shared similar characteristics. First, both inhibited IHVA in a dose-dependent manner and were reversed by the TRPV1 receptor antagonist capsazepine. Second, like capsaicin, high-concentration anandamide induced inward currents in cultured trigeminal ganglion neurons. Similar to capsaicin-induced inward current, the reversal potential of anandamide-induced current was also around 0 mV. Anandamide had different effects at low and high concentration via the activation of different receptors. By comparing the contribution of CB1 and TRPV1 activation on anandamide-induced increases in intracellular Ca2+ concentration, we conclude that anandamide-induced inhibition of high voltage-activated Ca2+ channels and an increase in intracellular Ca2+ concentration have the same dual effects as activation of the receptor pathway.
Inhibition of high voltage-activated Ca2+ channels and modulation of Ca2+ influx are important for neuronal activity and neurotransmitter release induced by endocannabinoids. However, evidence also suggests that cannabinoids can couple to Gs[18] and Gq[19] and activate TRPV1 receptors to increase Ca2+ influx. In addition, the response of cannabinoids on intracellular Ca2+ concentration differs across cell types[34]. To elucidate the complex mechanisms underlying the intracellular Ca2+ response, we further tested the effect of anandamide on intracellular Ca2+ concentration. We found that anandamide increased intracellular Ca2+ and simultaneously inhibited IHVA in the same population of trigeminal ganglion neurons, providing strong evidence that an increase in the intracellular Ca2+ concentration is not due to IHVA. We found that anandamide had dual effects on the inhibition of IHVA and elevation of intracellular Ca2+ concentration. However, by comparing the contribution of second messenger system, we found that cAMP- and cGMP-dependent protein kinase antagonists both reversed the anandamide-induced increase in intracellular calcium concentration and IHVA inhibition. Application of a protein kinase C antagonist reversed the inhibition of IHVA but the anandamide-induced increase in intracellular Ca2+ concentration was not blocked. From this, we can conclude that the increase in intracellular Ca2+ concentration and the IHVA inhibition observed with anandamide are separate processes that share the same receptors but differ in intracellular signal transduction pathways.
CB1 receptor activation by endocannabinoids has been mostly reported to cause depression of neuronal excitability and neurotransmitter release in presynaptic primary neurons[101325283536]. Accumulating evidence indicates that cannabinoids may contribute to the potentiation of neurotransmission. Endocannabinoid release potentiates synaptic transmission via CB1 receptor activation and dopamine release in the goldfish Mauthner cell[37]. Moreover, endocannabinoids may potentiate hippocampal synaptic transmission via astrocytic CB1 activation[30]. In the suprachiasmatic nucleus, CB1 receptor activation can increase excitability of circadian clock neurons[38]. It is widely accepted that CB1 receptor activation suppresses neurotransmission by inhibiting high voltage-activated Ca2+ channels, and decreases Ca2+ influx. In contrast to this general hypothesis, we found that endocannabinoids caused elevation of the intracellular Ca2+ concentration in trigeminal ganglion neurons, which was triggered by endocannabinoid-evoked inward current around the resting membrane potential (−60 mV). Furthermore, endocannabinoid-induced increase in intracellular Ca2+ concentration and decrease of high voltage-activated Ca2+ channels were two independent events, which had the same dual effects via similar receptor pathways, but differed in the second messenger transduction pathway. Thus, our results suggest that presynaptic endocannabinoid release might potentiate neurotransmission via endocannabinoid-evoked current, increased Ca2+ influx, and increased intracellular Ca2+ concentration.
In summary, anandamide dose-dependently causes an increase in intracellular Ca2+ concentration, mediated by Ca2+ influx via anandamide-evoked currents, but not high voltage-activated Ca2+ channels. In the same population of trigeminal ganglion neurons, CB1 receptor activation-induced inhibition of IHVA and increase in high voltage-activated Ca2+ channels are two separate processes that share the same (CB1) receptors, but differ in signal transduction pathway. Thus, the effects of anandamide on high voltage-activated Ca2+ channels ultimately depend on the balance between the increased Ca2+ influx via anandamide-evoked currents and inhibition of high voltage-activated Ca2+ channels, suggesting a complex role of anandamide on inhibitory or excitatory neuromodulation. Contrary to the general hypothesis of endocannabinoid depression on neurotransmission, this study elucidates possible mechanisms underlying endocannabinoid-induced potentiation of neurotransmission via Ca2+ signaling modulation.
Materials and Methods
Design
A cytological in vitro study.
Time and setting
All data were collected at the Departments of Pharmacology and Physiology, Tongji Medical College, Huazhong University of Science and Technology in China from September 2003 to April 2008. Data analysis and manuscript writing were performed at Baylor Medical College, USA, from April 2011 to October 2012.
Materials
180 male Sprague-Dawley rats weighing 180–200 g and aged 6–8 weeks were used. All animal protocols were approved by the faculty of Laboratory Animal Science, Huazhong University of Science and Technology (license No. SYXK (E) 2009-0049). All the experiments followed the Guidance Suggestions for the Care and Use of Laboratory Animals, formulated by the Ministry of Science and Technology of China[39].
Methods
Cell dissociation
Trigeminal ganglion neurons from Sprague-Dawley rats were cultured as described previously[40]. Briefly, trigeminal ganglia were dissected aseptically and washed with cold (4°C) modified Hank's balanced salt solution containing NaCl 130 mmol/L, KCl 5 mmol/L, KH2 PO4 0.3 mmol/L, NaHCO3 4.0 mmol/L, NaH2 PO4 0.3 mmol/L, D-glucose 5.6 mmol/L, and ethylene glycol bis(alpha-aminoethyl ether)-N, N′-tetraacetic acid 10 mmol/L, hydroxyethyl piperazine ethanesulfonic acid 10 mmol/L, at pH 7.4. The ganglia were chopped into small pieces, and then incubated in 3 mL modified Hank's solution with 0.1% collagenase (type XI–S) for 20–40 minutes at 37°C. Individual cells were dissociated by triturating them through a fire-polished glass pipette, followed by incubating with 10 μg/mL DNase I (type lV) in F12 medium (Life Technologies, Gaithersburg, MD, USA) for 10 minutes at 37°C, before centrifuging for 5 minutes at 1,000 r/min. After centrifuging three times, the cells were cultured in F12 supplemented with 10% fetal bovine serum. The cells were planted on poly-D-lysine pre-coated glass coverslips (15 mm diameter) and cultured no more than 12 hours at 37°C in a water saturated atmosphere with 5% CO2.
Patch-clamp recording
The cells were placed in a recording chamber mounted on an inverted microscope (Leica Inc., Solms, Germany) and perfused with extracellular solution at room temperature (21–22°C). Whole-cell patch-clamp experiments were carried out using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA, USA) and the output was digitized with a Digidata 1332A converter (Axon Instruments) and program pCLAMP 9.02 (Axon Instruments). Data were acquired at a sampling rate of 2 KHz. Cell membrane capacitance and series resistance were measured and compensated (> 90%). Data obtained from neurons in which uncompensated series resistance resulted in voltage-clamp errors > 5 mV were not taken into further analysis. The cell diameters were measured with a calibrated eyepiece under phase contrast illumination.
The resistance of the microelectrode was 2–4 MΩ when filled with the pipette solution. The microelectrode was made from G85150T-4 glass pipettes (Warner Instruments Inc., Hamden, CT, USA). The external solution contained Choline-Cl 110 mmol/L, TEACl 20 mmol/L, BaCl2 10 mmol/L, MgCl2 2.0 mmol/L, hydroxyethyl piperazine ethanesulfonic acid 10 mmol/L, and D-glucose 20 mmol/L, adjusted to pH 7.4 with CsOH. Ba2+ was used as the charge carrier when recording IHVA. Ba2+ was replaced by Ca2+ when recording anandamide-evoked currents. The pipette solution contained CsCl 120 mmol/L, CaCl2 0.1 mmol/L, MgCL2 2.0 mmol/L, ethylene glycol bis(alpha-aminoethyl ether)-N, N′-tetraacetic acid 10.0 mmol/L, hydroxyethyl piperazine ethanesulfonic acid 10.0 mmol/L and Tris-ATP 5.0 mmol/L, pH adjusted to 7.2 with CsOH. Small-sized cells were selected to perform the further experiments.
The volume of the recording chamber was about 1 mL and the local superfusion rate was 1 mL/min.
Calcium imaging
Drug-induced changes of high voltage-activated Ca2+ channels were measured using a confocal laser scanning imaging system (Fluoview FV500, Olympus, Tokyo, Japan). The trigeminal ganglion cells on glass cover slips were loaded with Fluo 2-AM by incubation with 1–5 μmol/L Fluo 2-AM in standard external solution and were maintained in the dark for 30–40 minutes. The standard external solution contained NaCL 140.0 mmol/L, KCL 5.0 mmol/L, CaCL2 2.0 mmol/L, MgCL2 1.0 mmol/L, glucose 10.0 mmol/L and hydroxyethyl piperazine ethanesulfonic acid 10.0 mmol/L, pH adjusted to 7.4 with NaOH. Ca2+ -free medium solution was identical except for the 2.0 mmol/L Ca2+ and additional 10 mmol/L ethyleneglycol bis(alpha-aminoethyl ether)-N, N′-tetraacetic acid to lower extracellular Ca2+ concentration. Small-sized trigeminal ganglion neurons (< 33 μmol/L) were selected to perform the further experiments.
Data analysis
The data were analyzed using pCLAMP 9.02 (Axon Instruments) and Sigmaplot 11.0 software (Systat Software Inc., San Jose, CA, USA). The amplitude of IHVA was calculated as the peak current. Voltage-dependent activation for the study of changes on IHVA was measured by a series of depolarized pulses (450 ms) from −50 mV to +40 mV, stepping by 10 mV with interval time of 5 seconds, at a holding potential of −80 mV. We fitted a Boltzmann function to the voltage-dependent activation curves, that is G/Gmax =1/[1+exp (V0.5–Vm)/k], where Gmax is the maximum conductance, V0.5 is the membrane potential at which 50% of activation was observed, and k is the slope of the function. Voltage-dependent inactivation was measured by a two pulse protocol in which the precondition pulses (3 seconds) ranged from −80 to +20 in 10 mV increments; following test pulse (200 millsecords) was +10 mV with an internal time of 6 seconds. The Boltzmann function was also fitted to the h-infinity curve, that is, I/Imax=1/[1+exp(V0.5–Vm)/k], where V0.5 is the membrane potential at which 50% of inactivation was observed, and k is the slope of the function. The dose-response curve was fitted by the Hill equation, in which, Ipeak=Ipeakmax/[1+(IC50/C)n], with IC50 as the concentration producing 50% inhibition and n as the Hill coefficient.
Statistical analysis
Data were presented as mean ± SEM. For all experiments, data were examined for Gaussian distribution first, and then analyzed for statistical significance using the paired or unpaired t-test and one-way analysis of variance by using Sigma plot 11.0 software (Systat Software Inc.). A value of P < 0.05 was considered statistically significant.