Cannabidiol Modulates M-Type K+ and Hyperpolarization-Activated Cation Currents

Cannabidiol (CBD) is a naturally occurring compound found in the Cannabis plant that is known for its potential therapeutic effects. However, its impact on membrane ionic currents remains a topic of debate. This study aimed to investigate how CBD modifies various types of ionic currents in pituitary GH3 cells. Results showed that exposure to CBD led to a concentration-dependent decrease in M-type K+ currents (IK(M)), with an IC50 of 3.6 μM, and caused the quasi-steady-state activation curve of the current to shift to a more depolarized potential with no changes in the curve’s steepness. The CBD-mediated block of IK(M) was not reversed by naloxone, suggesting that it was not mediated by opioid receptors. The IK(M) elicited by pulse-train stimulation was also decreased upon exposure to CBD. The magnitude of erg-mediated K+ currents was slightly reduced by adding CBD (10 μM), while the density of voltage-gated Na+ currents elicited by a short depolarizing pulse was not affected by it. Additionally, CBD decreased the magnitude of hyperpolarization-activated cation currents (Ih) with an IC50 of 3.3 μM, and the decrease was reversed by oxaliplatin. The quasi-steady-state activation curve of Ih was shifted in the leftward direction with no changes in the slope factor of the curve. CBD also diminished the strength of voltage-dependent hysteresis on Ih elicited by upright isosceles-triangular ramp voltage. Collectively, these findings suggest that CBD’s modification of ionic currents presented herein is independent of cannabinoid or opioid receptors and may exert a significant impact on the functional activities of excitable cells occurring in vitro or in vivo.


Introduction
Cannabidiol (CBD) is a non-psychoactive cannabinoid derived from the Cannabis plant, known for its potential therapeutic effects.It is among over 100 cannabinoids present in the plant and has been shown to be effective in treating various medical conditions, such as epilepsy, bipolar disorder, inflammation, and cancer [1][2][3][4][5].Recent studies have demonstrated that CBD can modify the activity in the hypothalamic-pituitary-adrenal axis [6,7] and can modulate different types of transmembrane ionic currents in electrically excitable cells, including the voltage-gated Na + current (I Na ) and the M-type K + currents (I K(M) ) [5,8,9].Thus, further clarification is necessary to determine the effects of CBD or related compounds on ionic currents present in the membranes of electrically excitable cells.
The KCNQ2, KCNQ3, and KCNQ5 genes encode the core subunits of K V 7.2, K V 7.3, and K V 7.5 channels, respectively [10].These potassium (K + ) channels, when activated, generate the M-type K + current (I K(M) ), which is characterized by its low threshold voltage activation and by slow activation and deactivation properties [11,12].The modulation of I K(M) has gained significant recognition as an additional therapeutic approach for treating a range of neurological disorders characterized by excessive neuronal activity.These disorders encompass conditions like cognitive dysfunction, neuropathic pain, and epilepsy [10,[13][14][15].Furthermore, the magnitude of I K(M) is believed to regulate the availability of voltage-gated Na + (Na V ) channels during prolonged high-frequency firing [16].Previous studies have shown that CBD can modify the magnitude of I K(M) [9,17].However, it is still largely unclear whether and how exposure to CBD can perturb the magnitude or gating properties of I K(M) .
The hyperpolarization-activated cation current (I h ), also known as the "funny current" (I f ), plays a crucial role in regulating repetitive electrical activity in cardiac cells, various types of central neurons, and endocrine or neuroendocrine cells [11,[18][19][20][21].This ionic current exhibits unique characteristics, including slow voltage-dependent activation kinetics and a mixed Na + /K + current that flows inwardly, and it can be blocked by CsCl or ivabradine [20,22].Activation of I h may lead to depolarization of the resting potential, reaching the threshold required for generating or triggering an action potential.Consequently, it influences pacemaker activity and impulse propagation [22].Additionally, the slow kinetics of I h in response to prolonged hyperpolarization can result in long-lasting, activitydependent modulation of membrane excitability across various types of excitable cells [23].I h is mediated by channels encoded by members of the hyperpolarization-activated cyclicnucleotide-gated (HCN) gene family, and studies have demonstrated that the activity of these channels underlies the ionic mechanisms associated with both convulsive disorders and inflammatory pain disorders [24][25][26].
Therefore, based on the aforementioned information, our aim was to investigate the effects of CBD or other related compounds on perturbations in various ionic currents (such as I Na , I K(M) , I h , and I K(erg) ) in pituitary GH 3 cells.The findings from this study highlight the evidence showing that CBD can directly and effectively inhibit the magnitude of I K(M) and I h in a concentration-and voltage-dependent manner.Such suppression of ionic currents appears to be direct and is highly unlikely to be linked to its binding to cannabinoid receptors.
Unless stated otherwise, the cell culture materials, such as Ham's F-12 medium, Lglutamine, horse serum, and fetal calf serum, were obtained from HyClone TM (Thermo Fisher; Level Biotech, Tainan, Taiwan).All other chemicals and reagents, such as CdCl 2 , CsCl, CsOH, HEPES, and aspartic acid, were commercially available and of analytical reagent grade.

Preparation of Pituitary GH 3 Cells
GH 3 pituitary tumor cells, originally derived from ATCC [CCL-82 TM ] and obtained from the Bioresources Collection and Research Center (BCRC-60015) in Hsinchu, Taiwan, were cultured in Ham's F-12 medium supplemented with 15% (v/v) horse serum, 2.5% (v/v) fetal calf serum, and 2 mM L-glutamine in a humidified environment with 5% CO 2 /95% air [19].The verification of GH 3 cells was conducted by measuring the level of prolactin in the culture medium.Experiments were conducted 5 to 6 days after the cells had reached 60-80% confluence.

Electrophysiological Recordings Using Patch-Clamp Technique
The GH 3 cells were dispersed just before each measurement with care.Then, a small amount of the cell suspension was rapidly transferred to a custom-made chamber, where the cells were allowed to settle on the bottom surface.A DM-IL inverted microscope (Leica; Highrise Instrument, Taichung, Taiwan) was used to monitor cell size during the experiments.A custom-made chamber was tightly placed on the microscope's stage, and a video camera system with a magnification of 1500× was connected to the microscope.The cells that were examined were maintained in a bath of normal Tyrode's solution with a concentration of 1.8 mM CaCl 2 , at room temperature (20-25 • C).
The patch electrodes were meticulously fashioned from Kimax-51 capillaries with an outer diameter of 1.5-1.8mm (#34500; Kimble, Dogger, New Taipei City, Taiwan) using a two-stage PP-830 puller (Narishige; Taiwan Instrument, Tainan, Taiwan).The electrode tips were then fire-polished using an MF-83 microforge (Narishige).Typically, the filled electrodes had tip resistances ranging from 2 to 4 MΩ.For standard patch-clamp recordings in a modified whole-cell configuration [19,20], we employed an RK-400 patch amplifier (Bio-Logic, Claix, France).The cell capacitance was measured to be 34 ± 6 pA (n = 32).Junction potentials, which arise at the electrode tip due to differences in composition between the internal solution and the bath solution, were nullified shortly before establishing a seal formation.Junction potential corrections were subsequently applied to the whole-cell data.During the recording process, the signal output data (i.e., potential or current tracings) were acquired and stored online at a frequency of 10 kHz or higher kHz.This was achieved using an ASUSPRO-BN401 LG laptop computer (ASUS, Yuan-Dai, Tainan, Taiwan) equipped with a Digidata 1440A (Molecular Devices; Advanced Biotech, New Taipei City, Taiwan).The entire system was controlled by pClamp 10.6 software (Molecular Devices).

Analyses of Whole-Cell Recordings
We analyzed the concentration-response curves of CBD-induced inhibition on the density of I K(M) or I h in GH 3 cells.To elicit the slowly activating I K(M) , cells were placed in high-K + , Ca 2+ -free solution, and we applied a 1 s depolarizing pulse to −10 mV from a holding potential of −50 mV, as described previously [11].The current densities were measured at the end of the depolarizing pulse in the presence or absence of exposure to different CBD concentrations.To evoke I h , cells were bathed in Ca 2+ -free Tyrode's solution and subjected to a 2 s hyperpolarizing pulse to −110 mV from a holding potential of −40 mV.The I h density was measured with or without CBD at the end of the hyperpolarizing pulse.The concentration of CBD needed to inhibit 50% of the I K(M) or I h (i.e., IC 50 ) was approximated using a Hill function as follows: where [CBD] is the CBD concentration applied, IC 50 is the half-maximal concentration of CBD, n H is the Hill coefficient, and E max is the maximal decrease in I K(M) or I h caused by the CBD presence.
To analyze the steady activation curve of I K(M) or I h in the absence and presence of CBD, we fitted the data using a Boltzmann function.The Boltzmann equation is defined as: where G max is the maximal conductance of either I K(M) or I h acquired from the absence or presence of 3 µM CBD, and V and V 1/2 represent the membrane potential in mV and the half-point of the activation curve of I K(M) or I h , respectively.The slope factor of the activation of I K(M) or I h is represented by k.

Methods for Curve-Fitting and Statistical Analyses
Continuous curves were fitted to the experimental data using linear or nonlinear regression methods, such as exponential or sigmoidal function.The software tools used for this purpose included pClamp 10.6 software (Molecular Devices), 64-bit OriginPro 2022b software (OriginLab ® ; Scientific Formosa, Kaohsiung, Taiwan), and Microsoft Excel (part of the Microsoft 365 suite) (Microsoft, Redmond, WA, USA) with the "Solver" add-in function.
The experimental data obtained from the experiments comprised various types of ionic currents and were presented using the mean value with corresponding standard error of the mean (SEM).The sample size (n) denoted the number of cells from which data were gathered, and the SEM error bars were included in the plots.The Kolmogorov-Smirnov test for normality suggested that the data distribution was acceptable and could be assumed as normal.Paired or unpaired Student's t-tests between the two groups were applied.To assess differences between more than two groups, we used an analysis of variance (ANOVA-1 or ANOVA-2) with or without repeated measures, followed by a post-hoc Fisher's least significant difference test.Differences were considered statistically significant at a p value < 0.05 (* or ** in the figures denote significance).

Effect of Cannabidiol (CBD) on the M-Type K + Current (I K(M) ) Identified in Pituitary GH 3 Cells
In the initial whole-cell current recordings, we first investigated whether CBD had an effect on the density of I K(M) in these cells.To assess whether CBD caused any perturbations in I K(M) , we exposed the cells to a high-K + , Ca 2+ -free solution containing 1 µM tetrodotoxin (TTX).The measuring electrode was filled with a K + -enriched solution, and we then held the examined cell at the level of −50 mV, and a 1 s depolarizing pulse to −10 mV was applied to evoke I K(M) .Under the experimental conditions described above, the activation of I K(M) was indicated by a slowly activating time course in response to long-lasting step depolarization.As the reversal potential of K + ions was around 0 mV, the resulting I K(M) was a slowly activating inward K + current that directed the flow of K + ions into the interior of the cell [11,12].Of interest, we observed a progressive reduction in the density of I K(M) following a 1 s membrane depolarization from −50 to −10 mV.This reduction was observed one minute after cells were subjected to the increasing concentrations of CBD (Figure 1A).For instance, when cells were exposed to 1 or 3 µM CBD, the density of I K(M) measured at the end of a 1 s membrane depolarization decreased to 3.8 ± 0.4 pA/pF (n = 8, p < 0.05) or 1.9 ± 0.2 pA/pF (n = 8, p < 0.05), respectively, from a control value of 3.9 ± 0.4 pA/pF (n = 8).Following the cessation of CBD exposure and subsequent washout, the current density returned to 3.7 ± 0.3 pA/pF (n = 8).Additionally, Figure 1B displays the concentration-response curve for the CBD-induced inhibition of the density of I K(M) , which was constructed with an IC 50 value of 3.6 µM.The results reflect that exposing GH 3 cells to CBD can cause a concentration-dependent decrease in the density of I K(M) , indicating a depressant action.centration-response curve for the CBD-induced inhibition of the d was constructed with an IC50 value of 3.6 μM.The results reflect tha to CBD can cause a concentration-dependent decrease in the density depressant action.

Effect of CBD on the Current Density Versus Voltage Relationship and State Activation Curve of IK(M) in GH3 Cells
To further investigate the inhibitory effect of CBD on IK(M), we density versus voltage relationship of IK(M) with and without CBD ad lustrates the quasi-steady-state current density versus voltage rela tained during the control period and after exposure to 3 μM CBD i served a marked decrease in the density of IK(M) elicited by depolarizi at voltage levels ranging between −30 and −10 mV, upon the addition

Effect of CBD on the Current Density Versus Voltage Relationship and the Quasi-Steady-State Activation Curve of I K(M) in GH 3 Cells
To further investigate the inhibitory effect of CBD on I K(M) , we analyzed the current density versus voltage relationship of I K(M) with and without CBD addition.Figure 2A illustrates the quasi-steady-state current density versus voltage relationship of I K(M) obtained during the control period and after exposure to 3 µM CBD in these cells.We observed a marked decrease in the density of I K(M) elicited by depolarizing steps, particularly at voltage levels ranging between −30 and −10 mV, upon the addition of CBD.
Moreover, the quasi-steady-state activation curve of I K(M) in the absence and presence of 3 µM CBD was constructed and plotted in Figure 2B.The normalized conductance of I K(M) with or without the presence of CBD was fitted to a Boltzmann function using the method described in Section 2. In the control, V 1/2 = −17.5 ± 0.8 mV, k = 4.9 ± 0.5 (n = 7), while in the presence of 3 µM CBD, V 1/2 = −10.4± 0.8 mV, k = 4.9 ± 0.5 (n = 7).The data reflected that CBD not only decreased the maximal magnitude of I K(M) , but it also shifted the quasi-steady-state activation curve of I K(M) to more depolarized potential (i.e., in the rightward direction) by approximately 7 mV.However, we found no clear adjustments in the slope factor (k value or steepness) of the curve when exposed to CBD, suggesting that there was no change in the gating charge of the curve during its exposure.Alternatively, the CBD-induced inhibition of I K(M) in unclamped cells could depend on the pre-existing resting membrane.
To further investigate the inhibitory effect of CBD on IK(M), we analyzed the cur density versus voltage relationship of IK(M) with and without CBD addition.Figure 2A lustrates the quasi-steady-state current density versus voltage relationship of IK(M) tained during the control period and after exposure to 3 μM CBD in these cells.We served a marked decrease in the density of IK(M) elicited by depolarizing steps, particul at voltage levels ranging between −30 and −10 mV, upon the addition of CBD.In another series of whole-cell experiments, we tested the possible effects of CBD, CBD plus naloxone, Lino, TRH, and Lira on the density of I K(M) elicited by membrane depolarization from −50 to −10 mV.Lino has been demonstrated to inhibit I K(M) potently [27], while TRH can suppress the magnitude of I K(M) [11].Lira was known to be an agonist of glucagon-like peptide-1 (GLP-1) receptor [28,29].As demonstrated in Figure 3A,B, CBD at a concentration of 3 µM produced an inhibitory effect on the I K(M) density; however, a subsequent addition of 10 µM naloxone failed to counteract CBD-induced inhibition of I K(M) .Meanwhile, the presence of Lino (3 µM), TRH (1 µM), or Lira (1 µM) was effective at suppressing the magnitude of I K(M) (Figure 3B).Naloxone is known to block opioid receptors.Naloxone at a concentration of 30 µM also did not cause any effect on the CBD-induced decrease in I K(M) .Hence, similar to TRH, Lira can suppress I K(M) possibly through its binding to GLP-1 receptors in pituitary cells [28].However, further addition of naloxone exerted no effect on the CBD-mediated decrease in I K(M) , suggesting that the CBD effect on I K(M) is independent of binding to opioid receptors.

Modification by CBD of I K(M) Elicited by Pulse-Train (PT) Stimulation
Previous work has shown the capability of I K(M) to maintain the availability of voltagegated Na + (Na V ) channels during prolonged high-frequency firing [16].Therefore, we further examined whether the CBD presence may modify the extent of I K(M) during PT depolarizing stimuli from −50 to −10 mV in GH 3 cells.As depicted in Figure 4, a oneminute exposure to 3 µM CBD resulted in a reduction in the density of both activating and deactivating I K(M) during 1 s PT stimulation.For instance, the presence of CBD (3 µM) decreased the density of activating I K(M) from 7.0 ± 1.1 to 3.7 ± 0.6 pA/pF (n = 7, p < 0.05) and the density of deactivating I K(M) from 25.8 ± 3.8 to 7.7 ± 1.2 pA/pF (n = 7, p < 0.05).Subsequent to the washout of the CBD, the densities of activating and deactivating I K(M) were restored to 6.8 ± 0.9 (n = 6) and 24.9 ± 3.5 pA/pF (n = 6), respectively.The obtained data provided insights into the persistent effectiveness of CBD-induced inhibition of I K(M) even under conditions of high PT stimulation.Thus, it is plausible to consider that the presence of Na V channels during high-frequency firing in unclamped excitable cells may experience additional inhibition during CBD exposure, despite CBD's previously descried potential to suppress Na V channel activity [8].

Modification by CBD of IK(M) Elicited by Pulse-Train (PT) Stimulation
Previous work has shown the capability of IK(M) to maintain the availability of voltagegated Na + (NaV) channels during prolonged high-frequency firing [16].Therefore, we further examined whether the CBD presence may modify the extent of IK(M) during PT depolarizing stimuli from −50 to −10 mV in GH3 cells.As depicted in Figure 4, a one-minute exposure to 3 μM CBD resulted in a reduction in the density of both activating and deac- channels during high-frequency firing in unclamped excitable cells may experie tional inhibition during CBD exposure, despite CBD's previously descried po suppress NaV channel activity [8].

Mild Inhibitory Effect of CBD on the erg-Mediated K + Current (IK(erg)) in GH3 Cells
We continued by exploring whether CBD can exert any effect on another t currents (i.e., IK(erg)) enriched in these cells.Cells were kept in high-K + , Ca 2+ -fre and we filled up the measuring electrode with K + -enriched solution.To evoke held the tested cell at the level of −10 mV and a hyperpolarizing step to −90 duration of 1 s was applied to it.As shown in Figure 5, one minute after being e CBD (10 μM), the density of IK(erg) in response to membrane hyperpolarization w icantly decreased to 8.6 ± 1.1 pA/pF (n = 8, p < 0.05) from a control value of 11.2 ± (n = 8), while CBD at a concentration of 3 μM did not have a clear effect on IK(e elicited by membrane hyperpolarization.Following the removal of the 10 μM subsequent washout, the IK(erg) density returned to 11.0 ± 1.4 pA/pF (n = 8).More ing continued exposure to 10 μM CBD, a further addition of NS1643 (10 μM) wa of attenuating CBD-mediated inhibition of IK(erg) (10.9 ± 1.5 pA/pF, n = 8, p < 0.05 cells.NS1643 has been reported to stimulate IK(erg) [30].The results therefore ind exposure to CBD slightly suppressed the density of IK(erg) in GH3 cells.

Mild Inhibitory Effect of CBD on the erg-Mediated K + Current (I K(erg) ) in GH 3 Cells
We continued by exploring whether CBD can exert any effect on another type of K + currents (i.e., I K(erg) ) enriched in these cells.Cells were kept in high-K + , Ca 2+ -free solution and we filled up the measuring electrode with K + -enriched solution.To evoke I K(erg) , we held the tested cell at the level of −10 mV and a hyperpolarizing step to −90 mV for a duration of 1 s was applied to it.As shown in Figure 5, one minute after being exposed to CBD (10 µM), the density of I K(erg) in response to membrane hyperpolarization was significantly decreased to 8.6 ± 1.1 pA/pF (n = 8, p < 0.05) from a control value of 11.2 ± 1.5 pA/pF (n = 8), while CBD at a concentration of 3 µM did not have a clear effect on I K(erg) density elicited by membrane hyperpolarization.Following the removal of the 10 µM CBD and subsequent washout, the I K(erg) density returned to 11.0 ± 1.4 pA/pF (n = 8).Moreover, during continued exposure to 10 µM CBD, a further addition of NS1643 (10 µM) was capable of attenuating CBD-mediated inhibition of I K(erg) (10.9 ± 1.5 pA/pF, n = 8, p < 0.05) in these cells.NS1643 has been reported to stimulate I K(erg) [30].The results therefore indicate that exposure to CBD slightly suppressed the density of I K(erg) in GH 3 cells.

Failure of CBD Effect on Na + Current (INa) in GH3 Cells
CBD was recently reported to suppress the magnitude of NaV1.4-encodedINa [8].our study, we conducted additional experiments to investigate whether the can also modify INa in GH3 cells.As shown in Figure 6, in whole-cell current recor ings, the magnitude of INa elicited in response to a short depolarizing pulse to −10 from holding potential of −100 mV was robustly observed; however, the INa density remain unaltered during a 2 min continued exposure to 10 μM CBD.Similarly, cannabichromen a compound known to bind to cannabinoid receptor 1 [1], did not have any effect on t density of INa.Moreover, with continued presence of 10 μM CBD, the further addition ranolazine (Ran, 10 μM) was effective at suppressing INa density, while the addition either tefluthrin (Tef, 10 μM) or telmisartan (TEL, 10 μM) enhanced current density.R is an inhibitor of late INa [31], while Tef or TEL can stimulate INa effectively [32].Therefo unlike NaV1.4-encoded currents [8], the INa observed in GH3 cells appears to be resista to modulation by CBD.3.6.Failure of CBD Effect on Voltage-Gated Na + Current (I Na ) in GH 3 Cells CBD was recently reported to suppress the magnitude of Na V 1.4-encodedI Na [8].In our study, we conducted additional experiments to investigate whether the presence of CBD can also modify I Na in GH 3 cells.As shown in Figure 6, in whole-cell current recordings, the magnitude of I Na elicited in response to a short depolarizing pulse to −10 from a holding potential of −100 mV was robustly observed; however, the I Na density remained unaltered during a 2 min continued exposure to 10 µM CBD.Similarly, cannabichromene, a compound known to bind to cannabinoid receptor 1 [1], did not have any effect on the density of I Na .Moreover, with continued presence of 10 µM CBD, the further addition of ranolazine (Ran, 10 µM) was effective at suppressing I Na density, while the addition of either tefluthrin (Tef, 10 µM) or telmisartan (TEL, 10 µM) enhanced current density.Ran is an inhibitor of late I Na [31], while Tef or TEL can stimulate I Na effectively [32].Therefore, unlike Na V 1.4-encoded currents [8], the I Na observed in GH 3 cells appears to be resistant to modulation by CBD.

Effect of CBD on Hyperpolarization-Activated Cation Current (Ih) Measured in GH3 Cells
We continued to examine the perturbations caused by CBD in Ih in these cells.To investigate the effects of CBD on Ih, we performed experiments under specific conditions First, we placed the cells in Ca 2+ -free Tyrode's solution containing 0.5 mM CdCl2 and 1 μM TTX.We used CdCl2 and TTX to block voltage-gated Ca 2+ and Na + currents, respectively The measuring electrode was backfilled with K + -enriched internal solution to evoke Ih.As the whole-cell mode was established, the tested cell was maintained at the level of −40 mV in voltage-clamp mode, and the voltage steps were subsequently applied (2 s in duration to a series of voltages ranging between −110 and −20 mV in 10 mV increments.As demon strated in Figure 7A, one minute after cells were exposed to 3 μM CBD, the Ih density elicited by 2 s membrane hyperpolarization to −110 mV from a holding potential of −40 mV was decreased.However, even with continued exposure to 3 μM CBD, the subsequen addition of 10 μM naloxone failed to reverse the CBD-induced decrease in Ih density Moreover, the quasi-steady-state current remained largely unaltered during CBD expo sure.

Effect of CBD on Hyperpolarization-Activated Cation Current (I h ) Measured in GH 3 Cells
We continued to examine the perturbations caused by CBD in I h in these cells.To investigate the effects of CBD on I h , we performed experiments under specific conditions.First, we placed the cells in Ca 2+ -free Tyrode's solution containing 0.5 mM CdCl 2 and 1 µM TTX.We used CdCl 2 and TTX to block voltage-gated Ca 2+ and Na + currents, respectively.The measuring electrode was backfilled with K + -enriched internal solution to evoke I h .As the whole-cell mode was established, the tested cell was maintained at the level of −40 mV in voltage-clamp mode, and the voltage steps were subsequently applied (2 s in duration) to a series of voltages ranging between −110 and −20 mV in 10 mV increments.As demonstrated in Figure 7A, one minute after cells were exposed to 3 µM CBD, the I h density elicited by 2 s membrane hyperpolarization to −110 mV from a holding potential of −40 mV was decreased.However, even with continued exposure to 3 µM CBD, the subsequent addition of 10 µM naloxone failed to reverse the CBD-induced decrease in I h density.Moreover, the quasi-steady-state current remained largely unaltered during CBD exposure.Of interest, one minute after cell exposure to 3 μM CBD, the density of Ih evoked by the 2 s long step hyperpolarization progressively decreased (Figure 7B,C).For instance, at the levels of −100 and −110 mV, it was observed that following a one-minute exposure to 3 μM CBD, the density of Ih diminished to 4.6 ± 0.8 pA/pF (n = 7, p < 0.05) and 7.6 ± 1.1 pA/pF (n = 7, p < 0.05), respectively.These values were reduced from the control values of 9.7 ± 1.4 pA/pF (n = 7) and 15.6 ± 1.7 pA/pF (n = 7).When CBD was removed, the Ih density which was fit to the data using least-squares minimization.The statistical analyses for (C,D) were undertaken by ANOVA-2 for repeated measures, p (factor 1, groups among data taken at different levels of membrane potential) < 0.05, p (factor 2, groups between the absence and presence of CBD) < 0.05, p (interaction) < 0.05, followed by post-hoc Fisher's test, p < 0.05.(E) Concentration-response curve of CBD-induced inhibition of I h density (mean ± SEM; n = 8 for each point).Current densities in the presence of different CBD concentrations were taken at the end of a 2 s hyperpolarizing pulse from −40 to −110 mV.The smooth curve was generated using the least-squares method and fitted to the Hill equation, as detailed in Section 2.
Of interest, one minute after cell exposure to 3 µM CBD, the density of I h evoked by the 2 s long step hyperpolarization progressively decreased (Figure 7B,C).For instance, at the levels of −100 and −110 mV, it was observed that following a one-minute exposure to 3 µM CBD, the density of I h diminished to 4.6 ± 0.8 pA/pF (n = 7, p < 0.05) and 7.6 ± 1.1 pA/pF (n = 7, p < 0.05), respectively.These values were reduced from the control values of 9.7 ± 1.4 pA/pF (n = 7) and 15.6 ± 1.7 pA/pF (n = 7).When CBD was removed, the I h density at −100 and −110 mV returned to 9.3 ± 1.1 pA/pF (n = 7) and 15.2 ± 1.6 pA/pF (n = 7), respectively.Additionally, the activating time constant (τ act ) of I h activated by the 2 s hyperpolarizing step from −40 to −110 mV decreased to 1.81 ± 0.04 s (n = 7, p < 0.05) from a control value of 1.07 ± 0.03 s (n =7).Moreover, with continued exposure to 3 µM CBD, a further addition of naloxone (10 µM) failed to reverse the CBD-mediated decrease in I h density.
Figure 7D illustrates the quasi-steady-state activation curve of I h acquired in the control period and during exposure to 3 µM CBD.According to the least-squares minimization procedure, the best fitting parameters (i.e., V 1/2 and k) for such a steady-state activation curve of the current were convergently acquired with or without the addition of CBD.In the presence of 3 µM CBD, the value of V 1/2 for the activation curve of the current was −101 ± 4 mV (n = 7), which was significantly distinguishable from that in the control period, −89 ± 4 mV (n = 7, p < 0.05).However, the k values in the absence (7.2 ± 0.3, n = 7) and presence of 3 µM CBD (7.3 ± 0.3, n = 7, p > 0.05) did not differ significantly.The experimental results showed that during exposure to 3 µM CBD, the activation curve of I h was shifted toward more hyperpolarized potential with no change in the slope factor of the curve (i.e., the steepness of the activation curve).Therefore, it appears likely that no change in the gating charge of I h activation curve was altered by CBD exposure.The responsiveness of I h to CBD is influenced by varying levels of pre-existing resting membrane potential.
As illustrated in Figure 7E, the presence of various concentrations of CBD can suppress the density of I h in a concentration-dependent manner.By virtue of a non-linear leastsquares fit to the experimental data, the IC 50 value for the inhibitory effect of CBD on I h was 3.3 µM, a value that is similar to that needed to suppress I K(M) density described above.These results indicate that CBD can exercise a depressant action on I h elicited by membrane hyperpolarization in these cells.Previous work has demonstrated the effectiveness of I h 's Hys (V) strength in affecting either various patterns of bursting firing or action potential configuration in varying types of excitable cells [33,34].Therefore, we continued by determining whether and how the presence of CBD may adjust the I h strength activated in response to long-lasting triangular V ramp .In this series of experiments, in the control period (i.e., absence of CBD), we maintained the tested cell at the level of −40 mV, and an upsloping (forward) limb from −150 to −40 mV followed by a downsloping (backward) limb back to −150 mV (i.e., upright isosceles-triangular V ramp ) for a total duration of 2 s with a ramp speed of ±55 mV/s was thereafter applied to evoke I h 's hysteresis (Hys (V) ) (Figure 8A,B).In accordance with previous studies [21,33,34], the Hys (V) of I h in response to such double V ramp was robustly observed and sensitive to suppression by ivabradine (IVA), an inhibitor of I h [19,20,26].Of additional interest, upon cell exposure to 3 µM CBD, the strength of I h 's Hys (V) responding to both rising and falling limbs of double V ramp progressively became depressed (Figure 8C).For instance, as the triangular V ramp was applied, the value of ∆area (i.e., the difference in area enclosed by the curve in the forward and backward direction) for Hys (V) in the control period was 248 ± 52 mV•(pA/pF) (n = 7), while the ∆area value of I h in the presence of 3 µM CBD was significantly reduced to 142 ± 28 mV•(pA/pF) (n = 7, p < 0.05).Moreover, a subsequent addition of oxaliplatin (OXAL, 10 µM) effectively reversed the CBD-induced decrease in Hys (V) 's strength.OXAL has been reported to activate I h [25,35].Therefore, the results indicate that cell exposure to CBD can modify the magnitude and gating properties of I h observed in GH 3 cells.

Discussion
This study identified seven significant findings.(1) Cannabinoid (CBD) expo sulted in a concentration-dependent suppression of M-type K + current (IK(M)) in p GH3 cells, with an IC50 of 3.6 μM.(2) The presence of CBD caused a rightward shi quasi-steady-state activation curve of IK(M) without changes in the slope factor (i.e., of the curve.(3) The CBD-induced block of IK(M) was not reversible by further add naloxone.(4) CBD exposure also suppressed IK(M) elicited by pulse-train (PT) depo stimuli.(5) CBD slightly reduced the magnitude of erg-mediated K + current (IK(erg)) not affect voltage-gated Na + current (INa) in GH3 cells.(6) Cell exposure to CBD r in the inhibition of hyperpolarization-activated cation current (Ih) with an IC50 of (7) The quasi-steady-state activation curve of Ih was shifted to a more hyperpolari tential without changes in the curve's slope factor.Based on the findings of this s can be inferred that CBD exerts a significant impact on transmembrane ionic c particularly IK(M) and Ih.Assuming that these effects would occur in vivo, they wou influence the firing frequency of action potential generation in cells.Therefore, research is necessary to determine the possible mechanisms of action of CBD o

Discussion
This study identified seven significant findings.(1) Cannabinoid (CBD) exposure resulted in a concentration-dependent suppression of M-type K + current (I K(M) ) in pituitary GH 3 cells, with an IC 50 of 3.6 µM.(2) The presence of CBD caused a rightward shift in the quasi-steady-state activation curve of I K(M) without changes in the slope factor (i.e., k value) of the curve.(3) The CBD-induced block of I K(M) was not reversible by further addition of naloxone.(4) CBD exposure also suppressed I K(M) elicited by pulse-train (PT) depolarizing stimuli.(5) CBD slightly reduced the magnitude of erg-mediated K + current (I K(erg) ) but did not affect voltage-gated Na + current (I Na ) in GH 3 cells.(6) Cell exposure to CBD resulted in the inhibition of hyperpolarization-activated cation current (I h ) with an IC 50 of 3.3 µM.(7) The quasi-steady-state activation curve of I h was shifted to a more hyperpolarized potential without changes in the curve's slope factor.Based on the findings of this study, it can be inferred that CBD exerts a significant impact on transmembrane ionic currents, particularly I K(M) and I h .Assuming that these effects would occur in vivo, they would likely influence the firing frequency of action potential generation in cells.Therefore, further research is necessary to determine the possible mechanisms of action of CBD or other structurally similar compounds in the body, such as the anxiolytic effects [3,6,7].
Different to some extent from a previous study [17], the current study found that the presence of CBD could suppress the magnitude of I K(M) .The IC 50 value for CBD to inhibit the density of I K(M) in GH 3 cells was 3.6 µM.The quasi-steady-state activation curve of I K(M) in GH 3 cells shifted towards a more depolarized potential, without changes in the curve's steepness.Additionally, the CBD presence could suppress the I K(M) elicited by PT stimulation.These findings suggest that the responsiveness of I K(M) in unclamped cells is influenced by various confounding factors, including the level of the pre-existing resting membrane potential, CBD concentration, patterns of action potential firing, or combinations of these variables.
It needs to be mentioned that, concerning the steady-state activation curve of I K(M) , it is important to have a sufficiently long step duration to accurately assess the slope factor and V 1/2 values.This is essential for a more precise determination of the activation curve of this current, followed by the application of the Boltzmann equation to deduce V 1/2 and the slope factor, subsequently allowing to predict the gating charge value.However, in the cells we have investigated (such as GH 3 cells), when we employed a voltage-clamp protocol that exceeds 1 sec, we often observed the appearance of the inactivation process from other types of K + currents.This phenomenon, to a certain extent, led to confusion with the inactivation of other delayed-rectifier K + currents.The primary reason behind this is that GH 3 cells, in addition to I K(M) , still possess various types of delayed-rectifier K + currents.This is a common issue encountered when conducting experiments on native excitable cells.Using cells with KCNQx-encoded currents may help mitigate these complications.Nonetheless, exposure of CBD to GH 3 cells did suppress the strength of I K(M) and shift the quasi-steady-state activation curve of I K(M) to a more depolarized potential without affecting the curve's slope factor.
Previous research has shown that a train of depolarizing pulses can effectively alter the magnitude of I Na , which is a current that decays exponentially over time [16].More recently, it has been demonstrated that the magnitude of I K(M) can regulate the availability of Na V channels during prolonged high-frequency firing [16].In our study, we found that exposure of GH 3 cells to CBD suppressed the magnitude of I K(M) during PT depolarizing stimuli.As a result, the availability of Na V channels during sustained high-frequency firing may be reduced, leading to a decrease in reliable presynaptic spiking and synaptic transmission at high frequencies [9,10,14].
Our study observed that CBD did not significantly alter the magnitude of I Na in GH 3 cells, nor did cannabichromene, an agonist of cannabinoid receptor 1 [1].These results appear to differ from the findings of a previous study conducted by Huang et al. [8].They reported the suppression of the density of Na V 1.4 currents by CBD.The reason for this discrepancy is not yet understood; however, it is possible that Na V 1.4 currents affected by CBD [8] are primarily expressed in skeletal muscle cells rather than endocrine or neuroendocrine cells.It is thus pertinent to conduct further investigation to determine if the effects of CBD on I Na are specific to certain tissues.
Four mammalian isoforms of HCN, which are HCN1, HCN2, HCN3, and HCN4, make up the macroscopic I h (or I f ) [22,33].Among them, HCN2, HCN3, and a combination of HCN2 and HCN3 channels are highly expressed in GH 3 cells and other types of endocrine or neuroendocrine cells [36].In our study, we observed that CBD caused a suppressive effect on I h in a concentration-and voltage-dependent manner.The IC 50 value for CBD to suppress I h was estimated as 3.3 µM, and the quasi-steady-state activation curve of I h was shifted to a more hyperpolarizing potential with no changes in the curve's steepness.The CBD exposure also resulted in a considerable reduction in the strength of I h 's Hys (V) elicited by long-lasting triangular V ramp .The effect of CBD on this Hys (V) behavior would be linked to its effect on the gating mechanism of HCN channels [37,38].Therefore, apart from its effectiveness in inhibiting I K(M) , as detailed above, CBD can also induce changes in the magnitude, gating kinetics, and Hys (V) behavior of I h in a manner that may have pharmacological, therapeutic, or toxicological significance.It is worth noting that clinically achievable levels of CBD have been reported [39].

Figure 1 .
Figure 1.Effect of cannabidiol (CBD) on M-type K + current (IK(M)) identified In this set of experiments, we bathed cells in high-K + , Ca 2+ -free solution, wh rodotoxin (TTX), and the measuring electrode used was filled up with a K + Representative current traces acquired in the control period (a) (i.e., absen cell exposure to 1 μM CBD (b) or 3 μM CBD (c).The upper part indicates the applied.(B) Concentration-response curve of CBD-induced block of IK(M) d cells (mean ± SEM; n = 8 for each point).The sigmoidal line drawn represent the Hill equation, as described in Section 2. The IC50 values for the CBD-med was optimally estimated to be 3.6 μM.

Figure 1 .
Figure 1.Effect of cannabidiol (CBD) on M-type K + current (I K(M) ) identified in pituitary GH 3 cells.In this set of experiments, we bathed cells in high-K + , Ca 2+ -free solution, which contained 1 µM tetrodotoxin (TTX), and the measuring electrode used was filled up with a K + -enriched solution.(A) Representative current traces acquired in the control period (a) (i.e., absence of CBD) and during cell exposure to 1 µM CBD (b) or 3 µM CBD (c).The upper part indicates the voltage-clamp protocol applied.(B) Concentration-response curve of CBD-induced block of I K(M) density observed in GH 3 cells (mean ± SEM; n = 8 for each point).The sigmoidal line drawn represents the goodness-of-fit to the Hill equation, as described in Section 2. The IC 50 values for the CBD-mediated inhibition of I K(M) was optimally estimated to be 3.6 µM.

Figure 2 .
Figure 2. Effect of CBD on mean current density versus voltage relationship (A) and the quasi-steadystate activation curve (B) of I K(M) .The experimental procedures employed in these experiments were similar to those outlined in Figure 1.The examined cell was maintained at −50 mV and a series of command voltages ranging from −50 to 0 mV in 10 mV steps were applied to it.(A) Mean current density versus voltage relationship of I K(M) in the absence (black filled squares) and presence (red open squares) of 3 µM CBD (mean ± SEM; n = 7 for each point).Current density was measured at the end of each voltage step.(B) Quasi-steady-state activation curve of I K(M) in the control (black filled squares) and during exposure to 3 µM CBD (red open squares) (mean ± SEM; n = 7 for each point).The smooth lines described in Section 2 were optimally generated by fitting the Boltzmann equation for the activation curve of the current using a least-squares method.The statistical analyses in (A) and (B) were undertaken by ANOVA-2 for repeated measures, p (factor 1, groups among data taken at different levels of membrane potential) < 0.05, p (factor 2, groups between the absence and presence of CBD) < 0.05, p (interaction) < 0.05, followed by post-hoc Fisher's test, p < 0.05.Of note, exposure to 3 µM CBD resulted in a rightward shift of the activation curve of I K(M) with no change in the slope factor of the curve.

17 Figure 3 .
Figure 3.Comparison among the impact of CBD, CBD plus naloxone, linopirdine (Lino), thyrotropin-releasing hormone (TRH), and liraglutinide (Lira) on the observed density of IK(M) in GH3 cells.(A) Representative current traces acquired in the control period (a) and during exposure to 3 μM CBD (b) or 3 μM CBD plus 10 μM naloxone (c).The upper part shows the voltage-clamp protocol applied.(B) Summary bar graph showing effect of CBD, CBD plus naloxone, linopirdine, thyrotropin-releasing hormone, and liraglutinide on IK(M) in GH3 cells.Current density was measured at the end of 1 s depolarizing pulse from −50 to −10 mV.Each bar indicates the mean ± SEM (n = 7).Data analysis was performed by ANOVA-1 (p < 0.05).* Significantly different from control (p < 0.05).

Figure 3 .
Figure 3.Comparison among the impact of CBD, CBD plus naloxone, linopirdine (Lino), thyrotropinreleasing hormone (TRH), and liraglutinide (Lira) on the observed density of I K(M) in GH 3 cells.(A) Representative current traces acquired in the control period (a) and during exposure to 3 µM CBD (b) or 3 µM CBD plus 10 µM naloxone (c).The upper part shows the voltage-clamp protocol applied.(B) Summary bar graph showing effect of CBD, CBD plus naloxone, linopirdine, thyrotropin-releasing hormone, and liraglutinide on I K(M) in GH 3 cells.Current density was measured at the end of 1 s depolarizing pulse from −50 to −10 mV.Each bar indicates the mean ± SEM (n = 7).Data analysis was performed by ANOVA-1 (p < 0.05).* Significantly different from control (p < 0.05).

Figure 4 .
Figure 4. Impact of CBD on the activation of IK(M) through pulse-train (PT) stimulation in To conduct the experiment, cells were placed in a high-K + , Ca 2+ -free solution, and the PT s protocol involved a series of 40 depolarizing pulses lasting 20 ms each, applied at −10 mV intervals, for a total duration of 1 s.(A) Representative current traces are presented, de cordings acquired during the control period (upper trace in black) and in the presence of (lower trace in red).The top portion of the figure shows the applied voltage-clamp prot symbol indicates the activating IK(M), while ** represents the deactivating (or tail) compon obtained after returning to −50 mV.Summary bar graphs in (B,C) display the activating tivating densities of IK(M), respectively, in the absence and presence of 1 or 3 μM CBD.The presented as mean ± SEM, with each bar representing data from seven independent ex The activating density of IK(M) was measured at the end of the PT depolarizing pulses f −10 mV, while the deactivating density was measured following the return to −50 mV.Da in (B) and (C) were performed by ANOVA-1 (p < 0.05).The * symbol indicates statistical s when compared to the control group (p < 0.05), while ** denotes statistical significance w pared to the CBD (1 μM) alone group (p < 0.05).

Figure 4 .
Figure 4. Impact of CBD on the activation of I K(M) through pulse-train (PT) stimulation in GH 3 cells.To conduct the experiment, cells were placed in a high-K + , Ca 2+ -free solution, and the PT stimulation protocol involved a series of 40 depolarizing pulses lasting 20 ms each, applied at −10 mV with 5 ms intervals, for a total duration of 1 s.(A) Representative current traces are presented, depicting recordings acquired during the control period (upper trace in black) and in the presence of 3 µM CBD (lower trace in red).The top portion of the figure shows the applied voltage-clamp protocol.The * symbol indicates the activating I K(M) , while ** represents the deactivating (or tail) component of I K(M) obtained after returning to −50 mV.Summary bar graphs in (B,C) display the activating and deactivating densities of I K(M) , respectively, in the absence and presence of 1 or 3 µM CBD.The values are presented as mean ± SEM, with each bar representing data from seven independent experiments.The activating density of I K(M) was measured at the end of the PT depolarizing pulses from −50 to −10 mV, while the deactivating density was measured following the return to −50 mV.Data analyses in (B) and (C) were performed by ANOVA-1 (p < 0.05).The * symbol indicates statistical significance when compared to the control group (p < 0.05), while ** denotes statistical significance when compared to the CBD (1 µM) alone group (p < 0.05).

Figure 5 .
Figure 5. Mild inhibitory effect of CBD on erg-mediated K + current (IK(erg)) in GH3 cells.We plac cells in high-K + , Ca 2+ -free solution containing 1 μM TTX, and the measuring pipette was filled w a K + -enriched solution.(A) Representative current traces obtained in the control period (a, bla color), and during cell exposure to 10 μM CBD (b, red color) or to 10 μM CBD plus 10 μM NS16 (c, green color).The voltage protocol applied is illustrated in the upper part.(B) Summary bar gra demonstrating effects of CBD (3 or 10 μM) and 10 μM CBD plus 10 μM NS1643 on the density IK(erg) (mean ± SEM; n = 8 for each bar).Current density (i.e., deactivating IK(erg)) was measured at t beginning of 1 s step hyperpolarization from −10 to −90 mV.Data analysis was performed ANOVA-1 (p < 0.05).* Significantly different from control (p < 0.05) and ** significantly differe from CBD (10 μM) alone group (p < 0.05).

Figure 5 .
Figure 5. Mild inhibitory effect of CBD on erg-mediated K + current (I K(erg) ) in GH 3 cells.We placed cells in high-K + , Ca 2+ -free solution containing 1 µM TTX, and the measuring pipette was filled with a K + -enriched solution.(A) Representative current traces obtained in the control period (a, black color), and during cell exposure to 10 µM CBD (b, red color) or to 10 µM CBD plus 10 µM NS1643 (c, green color).The voltage protocol applied is illustrated in the upper part.(B) Summary bar graph demonstrating effects of CBD (3 or 10 µM) and 10 µM CBD plus 10 µM NS1643 on the density of I K(erg) (mean ± SEM; n = 8 for each bar).Current density (i.e., deactivating I K(erg) ) was measured at the beginning of 1 s step hyperpolarization from −10 to −90 mV.Data analysis was performed by ANOVA-1 (p < 0.05).* Significantly different from control (p < 0.05) and ** significantly different from CBD (10 µM) alone group (p < 0.05).

Figure 6 .
Figure 6.Inability of CBD to modify the density of voltage-gated Na + current (INa) in GH3 cells.In these experiments, we placed cells in Ca 2+ -free Tyrode's solution that contained 10 mM tetrae thylammonium chloride, and the recording electrode was filled with a Cs + -enriched solution.(A Representative current traces acquired in the control period (a, black color) and during cell exposure to either 10 μM CBD alone (b, blue color) or 10 μM CBD plus 10 μM ranolazine (Ran) (c, red color) The voltage-clamp protocol applied is indicated in the upper part.(B) Summary bar graph demon strating effects of CBD, CBD plus ranolazine, CBD plus tefluthrin (Tef), and CBD plus telmisartan (TEL) on the peak density of INa (mean ± SEM; n = 7 for each bar).Of note, the presence of CBD (10 μM) did not cause any effect on the peak INa; however, further addition of Ran decreased INa density while that of either Tef or TEL increased it.Data analysis was performed by ANOVA-1 (p < 0.05).Significantly different from control (p < 0.05) and ** significantly different from CBD (10 μM) alone group (p < 0.05).

Figure 6 .
Figure 6.Inability of CBD to modify the density of voltage-gated Na + current (I Na ) in GH 3 cells.In these experiments, we placed cells in Ca 2+ -free Tyrode's solution that contained 10 mM tetraethylammonium chloride, and the recording electrode was filled with a Cs + -enriched solution.(A) Representative current traces acquired in the control period (a, black color) and during cell exposure to either 10 µM CBD alone (b, blue color) or 10 µM CBD plus 10 µM ranolazine (Ran) (c, red color).The voltage-clamp protocol applied is indicated in the upper part.(B) Summary bar graph demonstrating effects of CBD, CBD plus ranolazine, CBD plus tefluthrin (Tef), and CBD plus telmisartan (TEL) on the peak density of I Na (mean ± SEM; n = 7 for each bar).Of note, the presence of CBD (10 µM) did not cause any effect on the peak I Na ; however, further addition of Ran decreased I Na density, while that of either Tef or TEL increased it.Data analysis was performed by ANOVA-1 (p < 0.05).* Significantly different from control (p < 0.05) and ** significantly different from CBD (10 µM) alone group (p < 0.05).

Figure 7 .
Figure 7. Inhibitory effect of CBD on mean current density versus voltage relationship of hyperpolarization-activated cation current (Ih) recorded in GH3 cells.In this set of measurements, cells were kept in Ca 2+ -free Tyrode's solution that contained 1 μM TTX, and the measuring electrode was filled up with a K + -enriched solution.(A) Representative current traces acquired in the control period (a) and during exposure to 3 μM CBD (b) or 3 μM CBD plus 10 μM naloxone (c).The upper part indicates the voltage-clamp protocol used.(B) Representative current traces obtained in the control period (upper part) and during the presence of 3 μM CBD (lower part).The uppermost part represents the voltage-clamp protocol used, while the different colors correspond to different evoked currents (shown in the figure below).(C) Mean current density versus voltage relationship of Ih acquired in the absence (black filled squares) and presence (red open squares) of Ih density (mean ± SEM; n = 7 for each point).Current density was measured at the end of each hyperpolarizing pulse for a duration of 2 s.Quasi-steady-state activation curve of Ih obtained in the control period (black filled squares) and during exposure to 3 μM CBD (red open squares) (mean ± SEM; n = 7 for each point).The sigmoid curve in the figure represents the Boltzmann equation (shown in Section 2), which was fit to the data using least-squares minimization.The statistical analyses for (C,D) were undertaken by ANOVA-2 for repeated measures, p (factor 1, groups among data taken at different levels of membrane potential) < 0.05, p (factor 2, groups between the absence and presence of CBD) < 0.05, p (interaction) < 0.05, followed by post-hoc Fisher's test, p < 0.05.(E) Concentration-response curve of CBD-induced inhibition of Ih density (mean ± SEM; n = 8 for each point).Current densities in the presence of different CBD concentrations were taken at the end of a 2 s hyperpolarizing pulse from −40 to −110 mV.The smooth curve was generated using the least-squares method and fitted to the Hill equation, as detailed in Section 2.

Figure 7 .
Figure 7. Inhibitory effect of CBD on mean current density versus voltage relationship of hyperpolarization-activated cation current (I h ) recorded in GH 3 cells.In this set of measurements, cells were kept in Ca 2+ -free Tyrode's solution that contained 1 µM TTX, and the measuring electrode was filled up with a K + -enriched solution.(A) Representative current traces acquired in the control period (a) and during exposure to 3 µM CBD (b) or 3 µM CBD plus 10 µM naloxone (c).The upper part indicates the voltage-clamp protocol used.(B) Representative current traces obtained in the control period (upper part) and during the presence of 3 µM CBD (lower part).The uppermost part represents the voltage-clamp protocol used, while the different colors correspond to different evoked currents (shown in the figure below).(C) Mean current density versus voltage relationship of I h acquired in the absence (black filled squares) and presence (red open squares) of I h density (mean ± SEM; n = 7 for each point).Current density was measured at the end of each hyperpolarizing pulse for a duration of 2 s. (D) Quasi-steady-state activation curve of I h obtained in the control period (black filled squares) and during exposure to 3 µM CBD (red open squares) (mean ± SEM; n = 7 for each point).The sigmoid curve in the figure represents the Boltzmann equation (shown in Section 2), which was fit to the data using least-squares minimization.The statistical analyses for (C,D) were undertaken by ANOVA-2 for repeated measures, p (factor 1, groups among data taken at different levels of membrane potential) < 0.05, p (factor 2, groups between the absence and presence of CBD) < 0.05, p (interaction) < 0.05, followed by post-hoc Fisher's test, p < 0.05.(E) Concentration-response curve of CBD-induced inhibition of I h density (mean ± SEM; n = 8 for each point).Current densities in the presence of different CBD concentrations were taken at the end of a 2 s hyperpolarizing pulse from −40 to −110 mV.The smooth curve was generated using the least-squares method and fitted to the Hill equation, as detailed in Section 2.

Figure 8 .
Figure 8. Impact of CBD on Ih elicited by long-lasting triangular ramp voltage (Vramp) in G (A) Representative current traces acquired without (a, black color) or with (b, red color) the of 3 μM CBD.The upper part indicates the Vramp protocol applied, while dashed arrows s ascending and descending limbs of Vramp as a function of time.(B) Representative curren voltage relationship (i.e., voltage-dependent hysteresis, Hys(V)) of Ih elicited by 2 s triangu with or without the addition of 3 μM CBD.The dashed arrow indicates current trajector passes over time during triangular Vramp.(C) Summary bar graph showing effects of iv (IVA, 3 μM), CBD (3 μM), and CBD (3 μM) plus oxaliplatin (OXAL, 10 μM) on the area (Δ Vramp-induced Hys(V) in Ih (mean ± SEM; n = 7 for each bar).The Δarea represents the area by the upward (ascending) and downward (descending) limbs of the Ih elicited using an triangular Vramp usually ranging between −50 and −130 mV.Data analysis was perfo ANOVA-1 (p < 0.05).* Significantly different from control (p < 0.05) and ** significantly from CBD (3 μM) alone group (p < 0.05).

Figure 8 .
Figure 8. Impact of CBD on I h elicited by long-lasting triangular ramp voltage (V ramp ) in GH 3 cells.(A) Representative current traces acquired without (a, black color) or with (b, red color) the presence of 3 µM CBD.The upper part indicates the V ramp protocol applied, while dashed arrows show the ascending and descending limbs of V ramp as a function of time.(B) Representative current versus voltage relationship (i.e., voltage-dependent hysteresis, Hys (V) ) of I h elicited by 2 s triangular V ramp with or without the addition of 3 µM CBD.The dashed arrow indicates current trajectory which passes over time during triangular V ramp .(C) Summary bar graph showing effects of ivabradine (IVA, 3 µM), CBD (3 µM), and CBD (3 µM) plus oxaliplatin (OXAL, 10 µM) on the area (∆area) of V ramp -induced Hys (V) in I h (mean ± SEM; n = 7 for each bar).The ∆area represents the area enclosed by the upward (ascending) and downward (descending) limbs of the I h elicited using an isosceles triangular V ramp usually ranging between −50 and −130 mV.Data analysis was performed by ANOVA-1 (p < 0.05).* Significantly different from control (p < 0.05) and ** significantly different from CBD (3 µM) alone group (p < 0.05).