International Journal of Molecular Sciences

Article Bisoprolol, Known to Be a Selective β1-Receptor Antagonist, Differentially but Directly Suppresses IK(M) and IK(erg) in Pituitary Cells and Hippocampal Neurons

Edmund Cheung So 1,2,3, Ning-Ping Foo 1,3,4, Shun Yao Ko 3 and Sheng-Nan Wu 5,6,*

1 Department of Anesthesia, An Nan Hospital, China Medical University, Tainan 70965, Taiwan; [email protected] (E.C.S.); [email protected] (N.-P.F.) 2 Department of Anesthesia, China Medical University, Taichung 40402, Taiwan 3 Graduate Institute of Medical Sciences, Chang Jung Christian University, Tainan 71101, Taiwan; [email protected] 4 Department of Emergency Medicine, An Nan Hospital, China Medical University, Tainan 70965, Taiwan 5 Institute of Basic Medical Sciences, National Cheng Kung University Medical College, Tainan 70101, Taiwan 6 Department of Physiology, National Cheng Kung University Medical College, Tainan 70101, Taiwan * Correspondence: [email protected]; Tel.: +886-6-2353535-5334; Fax: +886-6-2362780



Received: 7 January 2019; Accepted: 30 January 2019; Published: 2 February 2019


Abstract: Bisoprolol (BIS) is a selective antagonist of β1 adrenergic receptors. We examined the effects + + of BIS on M-type K currents (IK(M)) or erg-mediated K currents (IK(erg)) in pituitary GH3, R1220 cells, and hippocampal mHippoE-14 cells. As GH3 cells were exposed to BIS, amplitude of IK(M) was suppressed with an IC50 value of 1.21 µM. The BIS-induced suppression of IK(M) amplitude was not affected by addition of isoproterenol or ractopamine, but attenuated by flupirtine or ivabradine. + In cell-attached current, BIS decreased the open probability of M-type K (KM) channels, along with decreased mean opening time of the channel. BIS decreased IK(erg) amplitude with an IC50 value of 6.42 µM. Further addition of PD-118057 attenuated BIS-mediated inhibition of IK(erg). Under current-clamp conditions, BIS depolarization increased the firing of spontaneous action potentials in GH3 cells; addition of flupirtine, but not ractopamine, reversed BIS-induced firing rate. In R1220 cells, BIS suppressed IK(M); subsequent application of ML-213(Kv7.2 channel activator) reversed BIS-induced suppression of the current. In hippocampal mHippoE-14 neurons, BIS inhibited IK(M) to a greater extent compared to its depressant effect on IK(erg). This demonstrated that in pituitary cells and hippocampal neurons the presence of BIS is capable of directly and differentially suppressing IK(M) and IK(erg), despite its antagonism of β1-adrenergic receptors.

Keywords: bisoprolol; M-type K+ current; M-type K+ channel. erg-mediated K+ current; membrane potential; pituitary cell

1. Introduction Bisoprolol (zebeta®; BIS), a phenoxy-2-propanol derivative, is recognized as an oral synthetic selective blocker of β1-adrenoceptor with antioxidant activity which exerts a number of potentially beneficial pharmacological effects such as atrial fibrillation, heart failure and postural tachycardia syndrome [1–4]. Due to its lipophilic nature, it facilitates entry into brain tissue to produce regulatory actions on central neurons [5,6]. Previous reports have demonstrated that BIS could bind to β1-adrenergic receptors inherently existing in brain areas including the pituitary gland and hippocampus [7–11]. An earlier study has also reported the ability of BIS to elevate blood prolactin level [12].

Int. J. Mol. Sci. 2019, 20, 657; doi:10.3390/ijms20030657 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2019, 20, 657 2 of 20

Carvedilol or other blockers of β-adrenergic receptors were indeed previously shown to suppress + + the amplitude of delayed-rectifier K currents (IK(DR)) and of HERG-mediated K currents [13,14]. There is also evidence showing that BIS appears to modulate a variety of ion currents. For example, previous reports have shown that BIS was able to reverse the down-regulation in mRNA expression of Na+, hyperpolarization-activated cation (HCN) and small-conductance Ca2+-activated K+ (SK) channels in failing hearts [15,16]. Surprisingly, little information is available regarding the underlying mechanism of actions of BIS or other structurally similar compounds on ionic currents in endocrine cells, neuroendocrine cells, or neurons, despite their wide clinical usage [2]. The KCNQ2, KCNQ3, and KCNQ5 genes are known to encode the core subunits of KV7.2, KV7.3 + and KV7.5 channels, respectively. The increased activity of these K channels can generate the M-type + K current (IK(M)) which is characterized by the activation in response to low threshold voltage and, once activated, displays a slowly activating and deactivating property [17–20]. Targeting IK(M) is growingly recognized as an adjunctive regimen for the treatment of many neurological disorders associated with neuronal hyper-excitability, such as cognitive dysfunction, neuropathic pain and epilepsy [21–24]. + The erg-mediated K current (IK(erg)) is encoded by three different subfamily of genes KCNH giving rise to the pore-forming α-subunit of erg (or KV11) channels. This type of current is characterized by slow activation and fast inactivation kinetics. Because of its rapid inactivation kinetics, it can result in an inward rectification. The magnitude of these currents in endocrine or neuroendocrine cells can contribute to the maintenance of the resting potential, thereby influencing the discharge frequency of action potentials and the stimulus-secretion coupling in these cells (APs) [25–28]. Earlier reports in our laboratory have shown the ability of ranolazine, risperidone or methadone to suppress IK(erg) in pituitary GH3 cells [25,27,29] However, to what extent the presence of BIS exerts any actions on these types of ionic currents (e.g., IK(M) and IK(erg)) remains elusive. In this study, we wanted to determine the possible underlying mechanism of BIS actions on perturbation of different ionic currents (e.g., IK(M), IK(erg) and IK(DR)) in pituitary GH3 cells and hippocampal mHippoE-14 neurons. Findings from this present study highlight the evidence to show that BIS can directly and differentially inhibit IK(M) and IK(erg) in a concentration-dependent manner in pituitary cells and hippocampal neurons. Such suppression of ion currents appears to be direct and is highly unlikely to be linked to its antagonistic action on β1 adrenergic receptors.

2. Results

2.1. Expression of KCNC1 (KV3.1), KCNQ2 (KV7.2) and KCNQ3 (KV7.3) in Pituitary GH3 Cells + In pituitary GH3 cells, as the M-type K current (IK(M)) was to be carried by the product of KCNQ2/KCNQ3 (or KV7.2/KV7.3) genes, we examined the mRNA levels of these genes. Our RT-PCR analysis demonstrated the mRNA expression of KCNQ2/KCNQ3 in these cells (Figure1).

2.2. Effect of Bisoprolol (BIS) on IK(M) in Pituitary GH3 Cells In the first set of whole-cell current recordings, we bathed the cells in high-K+, Ca2+-free Tyrode’s solution, the composition of which is described above, and the recording pipette was filled with K+-containing solution. Once the whole-cell model was established, the examined cell was maintained at −50 mV and the depolarizing step to −10 mV with a duration of 1 sec was delivered. As shown in Figure2, upon long-lasting membrane depolarization to −10 mV, K+ inward current was readily evoked, which was responsive to low threshold voltage and displayed both the slowly activating and deactivating properties. This type of K+ currents in response to depolarizing step was sensitive to inhibition by linopirdine (10 µM) or pioglitazone (10 µM), and it was hence identified as an IK(M) [17–20]. Of particular interest is how as GH3 cells were exposed to BIS, the IK(M) amplitude in response to such depolarizing step was progressively diminished in a concentration-dependent manner (Figure2A,B). For example, addition of 1-µM BIS significantly decreased current amplitude from 196 ± 14 to 97 ± 11 Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 2 of 20

Carvedilol or other blockers of β-adrenergic receptors were indeed previously shown to suppress the amplitude of delayed-rectifier K+ currents (IK(DR)) and of HERG-mediated K+ currents [13,14]. There is also evidence showing that BIS appears to modulate a variety of ion currents. For example, previous reports have shown that BIS was able to reverse the down-regulation in mRNA expression of Na+, hyperpolarization-activated cation (HCN) and small-conductance Ca2+-activated K+ (SK) channels in failing hearts [15,16]. Surprisingly, little information is available regarding the underlying mechanism of actions of BIS or other structurally similar compounds on ionic currents in endocrine cells, neuroendocrine cells, or neurons, despite their wide clinical usage [2]. The KCNQ2, KCNQ3, and KCNQ5 genes are known to encode the core subunits of KV7.2, KV7.3 and KV7.5 channels, respectively. The increased activity of these K+ channels can generate the M-type K+ current (IK(M)) which is characterized by the activation in response to low threshold voltage and, once activated, displays a slowly activating and deactivating property [17–20]. Targeting IK(M) is growingly recognized as an adjunctive regimen for the treatment of many neurological disorders associated with neuronal hyper-excitability, such as cognitive dysfunction, neuropathic pain and epilepsy [21–24]. The erg-mediated K+ current (IK(erg)) is encoded by three different subfamily of genes KCNH giving rise to the pore-forming α-subunit of erg (or KV11) channels. This type of current is characterized by slow activation and fast inactivation kinetics. Because of its rapid inactivation kinetics, it can result in an inward rectification. The magnitude of these currents in endocrine or neuroendocrine cells can contribute to the maintenance of the resting potential, thereby influencing the discharge frequency of action potentials and the stimulus-secretion coupling in these cells (APs) [25–28]. Earlier reports in our laboratory have shown the ability of ranolazine, risperidone or methadone to suppress IK(erg) in pituitary GH3 cells [25,27,29] However, to what extent the presence of BIS exerts any actions on these types of ionic currents (e.g., IK(M) and IK(erg)) remains elusive. In this study, we wanted to determine the possible underlying mechanism of BIS actions on perturbation of different ionic currents (e.g., IK(M), IK(erg) and IK(DR)) in pituitary GH3 cells and hippocampal mHippoE-14 neurons. Findings from this present study highlight the evidence to show that BIS can directly and differentially inhibit IK(M) and IK(erg) in a concentration-dependent manner in pituitary cells and hippocampal neurons. Such suppression of ion currents appears to be direct and is highly unlikely to be linked to its antagonistic action on β1 adrenergic receptors. Int. J. Mol. Sci. 2019, 20, 657 3 of 20 2. Results pA2.1. ( nExpression= 12, p < 0.05).of KCNC1 After (KV3.1), washout KCNQ2 of the (KV7.2) agent, and current KCNQ3 amplitude (KV7.3) in Pituitary returned GH to3 cells 191 ± 14 pA (n = 11, p < 0.05). Concomitant with this, the value of activation time constant (τact) during the exposure to In pituitary GH3 cells, as the M-type K+ current (IK(M)) was to be carried by the product of 1-µM BIS was noted to increase significantly from 102 ± 9 to 182 ± 11 msec (n = 12, p < 0.05) and that KCNQ2/KCNQ3Int. J. Mol. Sci. 2019 (or, 20 , Kx VFOR7.2/K PEERV7.3) REVIEW genes, we examined the mRNA levels of these genes. Our 3 of RT-PCR 20 of deactivating time constant (τ ) was decreased from 168 ± 11 to 66 ± 8 msec (n = 12, p < 0.05). analysis demonstrated the mRNAdeact expression of KCNQ2/KCNQ3 in these cells (Figure 1). products were obtained using for a marker lane of DNA molecular size (leftmost) and KCNQ2/3 subunit (198 bp). The labelling in the left side indicates the molecular weight.

2.2. Effect of Bisoprolol (BIS) on IK(M) in Pituitary GH3 Cells

In the first set of whole-cell current recordings, we bathed the cells in high-K+, Ca2+-free Tyrode’s solution, the composition of which is described above, and the recording pipette was filled with K+- containing solution. Once the whole-cell model was established, the examined cell was maintained at −50 mV and the depolarizing step to −10 mV with a duration of 1 sec was delivered. As shown in Figure 2, upon long-lasting membrane depolarization to −10 mV, K+ inward current was readily evoked, which was responsive to low threshold voltage and displayed both the slowly activating and deactivating properties. This type of K+ currents in response to depolarizing step was sensitive to inhibition by linopirdine (10 μM) or pioglitazone (10 μM), and it was hence identified as an IK(M) [17– 20]. Of particular interest is how as GH3 cells were exposed to BIS, the IK(M) amplitude in response to such depolarizing step was progressively diminished in a concentration-dependent manner (Figure Figure 1. The expression levels of the β-actin, KCNC1 (KV3.1), KCNQ2 (KV7.2) and KCNQ3 (KV7.3) 2AFigure and 2B).1. The For expression example, additionlevels of of the 1- μβM-actin, BIS significantlyKCNC1 (KV3.1), decreased KCNQ2 current (KV7.2) amplitude and KCNQ3 from (K 196V7.3) ± isolated14 to 97 from± 11 pA GH (n3 cells.= 12, p Total < 0.05). RNA After was washout isolated of and the RT-PCR agent, analysiscurrent amplitude was made. returned Amplified to 191 RT-PCR ± 14 isolated from GH3 cells. Total RNA was isolated and RT-PCR analysis was made. Amplified RT-PCR productspA (n = 11, were p < obtained0.05). Concomitant using for awith marker this, lane the ofvalue DNA of molecularactivation sizetime (leftmost)constant ( andτact) during KCNQ2/3 the subunitexposure (198 to 1- bp).μM The BIS labelling was noted in theto increase left side significantly indicates the from molecular 102 ± weight.9 to 182 ± 11 msec (n = 12, p <

0.05) and that of deactivating time constant (τdeact) was decreased from 168 ± 11 to 66 ± 8 msec (n = 12, Thep < 0.05). relationship between the BIS concentration (0.01–30 µM) and the IK(M) amplitude was further constructedThe relationship and between is plotted the inBIS Figure concentration2B. Fitting (0.01-30 the μ concentration-responseM) and the IK(M) amplitude curvewas further with the modifiedconstructed Hill equation and is plotted as described in Figure in2B. Materials Fitting the andconcentration-response Methods for details curve yielded with the a half-maximalmodified concentrationHill equation (i.e., as ICdescribed50) of 1.21 in MaterialsµM and and a slopeMethod coefficients for details of yielded 1.2. BIS a half-maximal at a concentration concentration of 30 µM nearly(i.e., abolished IC50) of 1.21 current μM and amplitude. a slope coefficient Additionally, of 1.2. BIS in at the a concentration continued presence of 30 μM of nearly 1-µM abolished BIS, further additioncurrent of eitheramplitude. 10 µ Additionally,M flupirtine in or the 10 continuedµM ivabradine presencewas of 1-μ notedM BIS, to further attenuate addition its suppressionof either 10 of μM flupirtine or 10 μM ivabradine was noted to attenuate its suppression of IK(M) (Figure 2C). IK(M) (Figure2C). Flupirtine was reported to stimulate IK(M) [30], while ivabradine known to suppress Flupirtine was reported to stimulate IK(M) [30], while ivabradine known to suppress hyperpolarization-activated cation current might increase I amplitude in these cells. Conversely, hyperpolarization-activated cation current might increase IK(M)K(M) amplitude in these cells. Conversely, subsequentsubsequent application application of neither of neither isoproterenol isoproterenol (1 µM) (1 nor μM) ractopamine nor ractopamine (10 µM) (10 had μ anyM) perturbationshad any on BIS-inducedperturbations block on ofBIS-inducedIK(M). Isoproterenol block of or ractopamineIK(M). Isoproterenol (a synthetic or ractopamine phenoethanolamine (a synthetic salt) was previouslyphenoethanolamine demonstrated salt) to was stimulate previouslyβ-adrenergic demonstrated receptors to stimulate [31–35 β-adrenergic]. However, receptors addition [31–35]. of neither isoproterenolHowever, (1 additionµM) nor of ractopamineneither isoproterenol (10 µM) (1 alone μM) producednor ractopamine any effects (10 μM) on IaloneK(M). produced The results any led us to suggesteffects thaton IK(M) BIS. hasThe aresults significant led us depressantto suggest that action BIS onhasI K(M)a significantfunctionally depressant expressed action in on GH IK(M)3 cells, whichfunctionally appears to expressed be unlinked in GH to binding3 cells, which to β-adrenergic appears to receptors.be unlinked to binding to β-adrenergic receptors.

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+ + Figure 2.FigureEffect 2. ofEffect bisoprolol of bisoprolol (BIS) (BIS) in the in amplitudethe amplitude of M-typeof M-type K Kcurrent current ( I(K(M)IK(M))) in in GH GH3 cells.3 cells. Cells Cells were were bathedbathed in high-K in high-K+ solution+ solution and and the pipettethe pipette was was filled filled with with K K+-containing+-containing solution, solution, the the composition composition of of which iswhich detailed is detailed in Materials in Materials and and Methods. Methods. As As the the whole-cell whole-cell mode mode was established, established, the the depolarizing depolarizing pulse frompulse −from50 to−50− to10 −10 mV mV (indicated (indicated inin thethe Inset Inset of of [A)]) [A)]) was was delivered delivered to the to examined the examined cells. (A cells.) Original IK(M) traces in response to long-lasting membrane depolarization. a: control; b: 0.1-μM BIS; c: (A) Original IK(M) traces in response to long-lasting membrane depolarization. a: control; b: 0.1-µM BIS; 0.3-μM BIS; and d: 1-μM BIS. (B) Concentration-response curve for BIS-induced inhibition of IK(M) in c: 0.3-µM BIS; and d: 1-µM BIS. (B) Concentration-response curve for BIS-induced inhibition of IK(M) GH3 cells. The amplitude of IK(M) during cell exposure to different BIS concentrations (0.01-30 μM) was µ in GH3comparedcells. The with amplitude the control of I valueK(M) during(mean ± cellSEM; exposure n = 12 for to each different data point). BIS concentrations The sigmoidal smooth (0.01–30 line M) was comparedrepresents with a best the fit control to a modified value (mean Hill function± SEM; describedn = 12 for in eachMaterials data and point). Methods. The sigmoidal The values smooth for

line representsIC50, maximally a best fitinhibited to a modified percentage Hill of function IK(M), and described the Hill coefficient in Materials were and1.21μ Methods.M, 100 %, Theand values1.2, for IC50respectively., maximally (C inhibited) Summary percentage bar graph ofshowingIK(M), the and effects the Hill of BIS, coefficient BIS plus were flupirtine, 1.21µ andM, 100BIS %,plus and 1.2, respectively.ivabradine (onC) I SummaryK(M) amplitude bar (mean graph ± showingSEM; n = 9 the for effects each bar). of BIS,BIS: BIS1-μM plus BIS; flupirtine, Flu: 10 μM andflupirtine; BIS plus

ivabradineIVA: on 10 IμK(M)M ivabradine.amplitude *Significantly (mean ± SEM; differentn = 9 from for each control bar). (p BIS:< 0.05) 1-µ andM BIS; **significantly Flu: 10 µM different flupirtine; IVA: 10fromµM BIS ivabradine. (1 μM) alone * Significantly group (p < 0.05). different from control (p < 0.05) and ** significantly different from BIS (1 µM) alone group (p < 0.05). 2.3. Inhibitory Effect of BIS on IK(M) Elicited by High Frequency Depolarizing Pulse 2.3. InhibitorySome Effect neurons, of BIS neuroendocrine on IK(M) Elicited ce bylls, High or endocrine Frequency cells Depolarizing can display Pulse firing behavior with high Somefrequency neurons, [25,36,37]. neuroendocrine For this reason, cells, in oranother endocrine set of experiments, cells can display we sought firing to behaviorevaluate whether with high frequencyBIS affects [25,36 ,the37]. magnitude For this reason, of IK(M) elicited in another by brief set repetitive of experiments, depolarizations. we sought Under to evaluate control conditions, whether BIS a single 1-sec depolarizing step to −10 mV from a holding potential of −50 mV evoked IK(M) amplitude affects the magnitude of IK(M) elicited by brief repetitive depolarizations. Under control conditions, of 156 ± 16 pA (n = 13) with the τact value of 53 ± 7 msec (n = 13). However, the IK(M) amplitude for 1- − − a singlesec 1-sec repetitive depolarizing pulses to step −10 to mV,10 each mV of from which a holdinglasted 6 msec potential with of4-msec50 intervals mV evoked at theIK(M) levelamplitude of −50 of 156 ±mV16 between pA (n = each 13) abrupt with the pulseτact wavalues evidently of 53 reduced± 7 msec to 34 (n ±= 5 13). pA (n However, = 13), together the I K(M)with aamplitude significant for 1-sec repetitiveprolongation pulses of τact to value−10 to mV, 69 each± 8 msec of which(n = 13). lasted A representative 6 msec with example 4-msec of intervalsIK(M) traces atin theresponse level of −50 mVto either between single each pulse abrupt or repetitive pulse waspulses evidently is illustrated reduced in Figure to 343A.± As5 shown pA (n in= Figure 13), together 3B, addition with a significantof BIS prolongation was able to ofsuppressτact value the I toK(M) 69 amplitude± 8 msec elicited (n = 13). by Ahigh-frequency representative depolarizing example of pulse.IK(M) Fortraces in responseexample, to either as cells single were pulseexposed or to repetitive 3-μM BIS, pulses current is amplitude illustrated measured in Figure at3 A.the As end shown of a train in Figureof short3 B, repetitive stimuli was further reduced by 56 ± 2 % to 19 ± 3 pA (n = 13, p < 0.05). Therefore, IK(M) addition of BIS was able to suppress the IK(M) amplitude elicited by high-frequency depolarizing pulse. activation was reduced by repetitive stimuli, and the inhibition by this agent of IK(M) amplitude still For example, as cells were exposed to 3-µM BIS, current amplitude measured at the end of a train of remained effective. However, the magnitude of its block appears to become smaller, as compared to short repetitive stimuli was further reduced by 56 ± 2 % to 19 ± 3 pA (n = 13, p < 0.05). Therefore, those with a single long depolarizing pulse. IK(M) activation was reduced by repetitive stimuli, and the inhibition by this agent of IK(M) amplitude still remained effective. However, the magnitude of its block appears to become smaller, as compared to those with a single long depolarizing pulse.

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FigureFigure 3. 3. AccumulativeAccumulative activation activation of of IK(M)IK(M) duringduring repetitive repetitive stimuli stimuli obtained obtained in inthe the absence absence and and + 2+ presencepresence of of BIS BIS in in GH GH3 3cells.cells. Cells Cells were were bathed bathed in in high-K high-K+, ,Ca Ca2+-free-free solution. solution. (A) (A )Representative Representative currentcurrent trace trace in inresponse response to tosingle single step step or repetitive or repetitive pulses pulses (b). (b).In the In lower the lower part of part (a), of current (a), current was evokedwas evoked in response in response to single to singlemembrane membrane depolarization depolarization from− from50 to− −1050 tomV− with10 mV a duration with a duration of 1 sec of (indicated1 sec (indicated in the inupper the upperpart of part (a)), of (a)),whereas whereas that thatin (b) in (b)was was obtained obtained during during rapid rapid repetitive repetitive depolarizationsdepolarizations to to−10− mV10 mV (indicated (indicated in the in lower the lower part partof (b)). of The (b)). individual The individual depolarizing depolarizing pulse used pulse + toused elicit to inward elicit inwardK+ current K currentlasted 6 lastedmsec with 6 msec a total with duration a totalduration of 1 sec. ofArrowheads 1 sec. Arrowheads in (a) and in (b) (a)

indicateand (b) the indicate zero current the zero level, current while level, open while arrow open indicated arrow in indicated (b) depicts in the (b) level depicts of IK(M) the elicited. level of TheIK(M) activationelicited. Thetime activation constants timefor control constants IK(M) for traces control in responseIK(M) traces to single in response step and to singlerepetitive step depolarizing and repetitive pulsesdepolarizing were 53 pulsesand 69 were msec, 53 respectively. and 69 msec, (B) respectively. Summary bar (B) graph Summary showing bar graph the effects showing of BIS the (1 effects and 3 of µ I ± n μM)BIS on (1 andthe amplitude 3 M) on the of amplitudeIK(M) during of repetitiveK(M) during depo repetitivelarizations depolarizations (mean ± SEM; (mean n = 13 SEM;for each= bar). 13 for Currenteach bar). amplitude Current was amplitude measured was at measured the end atof thesingle end step of single or repetitive step or repetitive depolarizing depolarizing pulses (i.e., pulses 1 sec).(i.e., *or** 1 sec). indicates *or** indicates significantly significantly different different from the from controls the controls taken with taken single with singledepolarizing depolarizing step (p step < 0.05)(p < or 0.05) repetitive or repetitive stimuli stimuli (p < 0.05),(p < 0.05),respectively. respectively.

2.4. Effect of BIS on Deactivating IK(M) Elicited Upon Return to Membrane Hyperpolarization with 2.4.Varying Effect Durationof BIS on Deactivating IK(M) Elicited Upon Return to Membrane Hyperpolarization with Varying Duration Previous studies have shown that the magnitude of IK(M) might influence the falling phase of burstingPrevious firing studies or spike have after-depolarizations shown that the magnitude [18,38]. Thus, of IK(M) we might wanted influence to determine the falling how BIS phase exerted of bursting firing or spike after-depolarizations [18,38]. Thus, we wanted to determine how BIS exerted any perturbations on deactivating IK(M) in response to the down sloping ramp pulse from −10 to any−50 perturbations mV with varying on deactivating durations. IK(M) As in shown response in Figure to the4 downA and sloping 4B, upon ramp return pulse to from−50 − mV,10 to as −50 the mV with varying durations. As shown in Figure 4A and 4B, upon return to −50 mV, as the slope of slope of ramp pulse was slowed, the peak amplitude of deactivating IK(M) became exponentially rampdecreased pulse withwas slowed, a time constant the peak of amplitude 98 ± 8 msec of deactivating (n = 11). However, IK(M) became as cells exponentially were exposed decreased to 3-µM withBIS, a the time peak constant amplitude of 98 of± 8 the msec current (n = 11). was However, significantly as cells and were exponentially exposed to decreased, 3-μM BIS, with the apeak time amplitudeconstant ofof65 the± current7 msec was(n = significantly 11). For example, and expone as the durationntially decreased, of downslope with rampa time was constant set at of 40 65 msec ± 7 (i.e.,msecslope (n = 11). = − 1For V/sec example,), the additionas the duration of 3-µM of BIS downsl decreasedope ramp peak was amplitude set at 40 by msec 59 ± (i.e.,2 % slope from 607= −1± V/sec),50 to 245the ±addition36 pA (ofn =3- 11,μMp BIS< 0.05). decreased The results peak amplitude thus indicated by 59 that, ± 2 as% from the duration 607 ± 50 of to down 245 ± sloping36 pA (n = 11, p < 0.05). The results thus indicated that, as the duration of down sloping ramp pulse increased, ramp pulse increased, the amplitude of deactivating IK(M) would be exponentially decreased, and that thethe amplitude presence ofof BISdeactivating decreased I currentK(M) would magnitude be exponentially in a time-dependent decreased, fashion.and that the presence of BIS decreased current magnitude in a time-dependent fashion.

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FigureFigure 4. Effect 4. Effect of BIS of onBIS the on deactivating the deactivatingIK(M) IinK(M) response in response to depolarizing to depolarizing pulse pulse with with different different durationsdurations (10-280 (10-280 msec) msec) of falling of falling phase phase which which was usedwas us toed mimic to mimic different different repolarizing repolarizing slope slope of of burstingbursting pattern. pattern. (A) Superimposed(A) Superimposed current current traces traces in response in respon tose the to uppermost the uppermost voltage voltage protocol protocol obtainedobtained in the in absence the absence (upper) (upper) andpresence and presence (lower) (lower) of 10- ofµM 10- BIS.μM TheBIS. uppermost The uppermost part indicatespart indicates the the voltage profile applied. (B) Effect of BIS on deactivating I upon return to −50 mV with different voltage profile applied. (B) Effect of BIS on deactivatingK(M) IK(M) upon return to −50 mV with different durations (mean ± SEM; n = 11 for each data point). The peak amplitude of deactivating I was durations (mean ± SEM; n = 11 for each data point). The peak amplitude of deactivating IK(M)K(M) was taken µ takenat at different different durations durations of of falling falling phase. phase. ■: control;: control; □: in: inthe the presence presence of of 3- 3-μMM BIS. BIS. + 2.5. Inhibitory Effect of BIS on the Activity of Single M-type K (KM) Channels in GH3 Cells 2.5. Inhibitory Effect of BIS on the Activity of Single M-type K+ (KM) Channels in GH3 Cells The cell-attached configuration of the patch-clamp technique was used to investigate the effects The cell-attached configuration of the patch-clamp technique was used to investigate the effects of BIS on the activity of single KM channels expressed in these cells. In these experiments, we bathed of BIS on the activity of single KM channels expressed in these cells. In these experiments, we bathed cells in high-K+, Ca2+-free solution and the recording pipette was filled with low-K+ (5.4 mM) solution. cells in high-K+, Ca2+-free solution and the recording pipette was filled with low-K+ (5.4 mM) solution. As shown in Figure5A, the activity of K M channels, which occurred in rapid open-closed transitions, As shown in Figure 5A, the activity of KM channels, which occurred in rapid open-closed transitions, was clearly detected, as the membrane was maintained at 0 mV relative to the bath. The addition was clearly detected, as the membrane was maintained at 0 mV relative to the bath. The addition of of 1-µM BIS significantly decreased channel open probability from 0.129 ± 0.006 to 0.056 ± 0.003 1-μM BIS significantly decreased channel open probability from 0.129 ± 0.006 to 0.056 ± 0.003 (n = 12, (n = 12, p < 0.01). After washout of the drug, channel activity was returned to 0.114 ± 0.004 (n = 10, p < 0.01). After washout of the drug, channel activity was returned to 0.114 ± 0.004 (n = 10, p < 0.01). p < 0.01). Based on an amplitude histogram (Figure5B), the single-channel amplitude did not differ Based on an amplitude histogram (Figure 5B), the single-channel amplitude did not differ between between the absence and presence of 1-µM BIS. Moreover, in continued presence of 1 µM BIS, further the absence and presence of 1-μM BIS. Moreover, in continued presence of 1 μM BIS, further addition addition of neither diazoxide (10 µM), 9-phenenthrol (10 µM), nor GMQ (10 µM) attenuated its of neither diazoxide (10 μM), 9-phenenthrol (10 μM), nor GMQ (10 μM) attenuated its suppression suppression of channel activity. Diazoxide and 9-phenanthrol are the openers of ATP-sensitive K+ of channel activity. Diazoxide and 9-phenanthrol are the openers of ATP-sensitive K+ and and intermediate-conductance Ca2+-activated K+ channels [39], respectively, while GMQ can activate intermediate-conductance Ca2+-activated K+ channels [39], respectively, while GMQ can activate large-conductance Ca2+-activated K+ channels [40]. These results strongly indicate that the channel large-conductance Ca2+-activated K+ channels [40]. These results strongly indicate that the channel activity suppressed by the presence of BIS ascribes primarily from KM channels. activity suppressed by the presence of BIS ascribes primarily from KM channels.

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A B A B + FigureFigure 5. 5. EffectEffect of of BIS BIS on on single single channel channel activity activity of of M-type M-type K K+ (K(KMM) )channels channels in in GH GH3 3cells.cells. In In these these Figure 5. Effect of BIS on single channel activity of M-type K+ (KM) channels in GH3 cells. In these currentcurrent recordings, recordings, cells cells were were bathed bathed in in high-K high-K+,+ ,Ca Ca2+2+-free-free solution solution and and the the recording recording pipette pipette was was current recordings, cells were bathed in high-K+, Ca2+-free solution and the recording pipette was filledfilled with with low-K low-K+ +(5.4(5.4 mM) mM) solution, solution, the the composition composition of of which which is isdescribed described in in Materials Materials and and Methods. Methods. filled with low-K+ (5.4 mM) solution, the composition of which is described in Materials and Methods. (A(A) )Original Original single single K KMM channelschannels obtained obtained in in the the absence absence (upper) (upper) and and presence presence of of 1- 1-μMµM BIS BIS (lower). (lower). (A) Original single KM channels obtained in the absence (upper) and presence of 1-μM BIS (lower). TheThe potential potential was maintainedmaintained at at 0 mV0 mV relative relative to theto bath.the bath. The upwardThe upward deflection deflection indicates indicates the opening the The potential was maintained at 0 mV relative to the bath. The upward deflection indicates the openingevent of event the channel. of the channel. (B) Amplitude (B) Amplitude histogram histogram obtained obtained in the control in the (left) control and during(left) and the during exposure the to opening event of the channel. (B) Amplitude histogram obtained in the control (left) and during the exposure1-µM BIS to (right). 1-μM BIS The (right). smooth The line smooth shown line in each shown histogram in each histogram indicates the indicates Gaussian the curve. Gaussian curve. exposure to 1-μM BIS (right). The smooth line shown in each histogram indicates the Gaussian curve. 2.6. Effect of BIS on Mean Open Time of KM Channels in GH3 Cells 2.6. Effect of BIS on Mean Open Time of KM Channels in GH3 Cells 2.6. Effect of BIS on Mean Open Time of KM Channels in GH3 Cells After observing that during the exposure to BIS, the open-time duration of KM channels in After observing that during the exposure to BIS, the open-time duration of KM channels in GH3 GH tendedAfter observing to be shortened that during with the no exposure changein to single-channelBIS, the open-time amplitude duration (Figure of KM5 channelsA), we further in GH3 tended3 to be shortened with no change in single-channel amplitude (Figure 5A), we further examined examinedtended to be and shortened analyzed with the no kinetic change properties in single-c ofhannel KM channels amplitude obtained (Figure with 5A), orwe without further examined addition and analyzed the kinetic properties of KM channels obtained with or without addition of BIS. As ofand BIS. analyzed As depicted the kinetic in Figure properties6, the distributionof KM channels of open obtained durations with or was without least-squares addition fitted of BIS. by As a depicted in Figure 6, the distribution of open durations was least-squares fitted by a mono- mono-exponentialdepicted in Figure function. 6, the Duringdistribution the exposure of open to durations 1-µM BIS, was the meanleast-squares open time fitted of these by channelsa mono- exponential function. During the exposure to 1-μM BIS, the mean open time of these channels was wasexponential significantly function. reduced During to 1.67 the ±exposure0.05 msec to 1- fromμM theBIS, controlthe mean length open of time 2.32 of± these0.09 msecchannels (n = was 12, significantly reduced to 1.67 ± 0.05 msec from the control length of 2.32 ± 0.09 msec (n = 12, p < 0.05). psignificantly< 0.05). Therefore, reduced BIS-induced to 1.67 ± 0.05 changes msec infrom KM -channelthe control activity length may of 2.32 have ± been 0.09 amsec result (n of = the12, decreasep < 0.05). Therefore, BIS-induced changes in KM-channel activity may have been a result of the decrease in the inTherefore, the duration BIS-induced of the open changes state, ratherin KM-channel than of anyactivity modification may have in been single-channel a result of the conductance. decrease in the duration of the open state, rather than of any modification in single-channel conductance. duration of the open 30state, rather than of any modification30 in single-channel conductance. 30 30 control BIS control BIS

20 20 20 20 events events events 10 10events 10 10

0 0 0 0.1 1 10 0 0.1 1 10 0.1msec 1 10 0.1msec 1 10 msec msec

FigureFigure 6. 6. EffectEffect of of BIS BIS on on mean mean open open time time of of K KMM channelschannels recorded recorded from from GH GH3 3cells.cells. In In control, control, data data Figure 6. Effect of BIS on mean open time of KM channels recorded from GH3 cells. In control, data werewere obtained obtained from from measurements measurements of of 480 480 channel channel op openings,enings, with with a atotal total recording recording time time of of 2 2min, min, were obtained from measurements of 480 channel openings, with a total recording time of 2 min, whereaswhereas in in the the presence presence of 1-1-µμMM BIS,BIS, datadata were were from from 371 371 channel channel openings, openings, with with a total a total recording recording time whereas in the presence of 1-μM BIS, data were from 371 channel openings, with a total recording√ timeof 3 min.of 3 min. Note Note that the that ordinate the ordinate and abscissa and abscissa indicate theindicate square the root square of the root event of number the event (i.e., numberevent ) time of 3 min. Note that the ordinate and abscissa indicate the square root of the event number (i.e.,and√event the logarithm) and the logarithm of open time of open (msec) time and, (msec) respectively. and, respectively. The smooth The linesmoot shownh line inshown each in lifetime each (i.e.,√event) and the logarithm of open time (msec) and, respectively. The smooth line shown in each lifetimedistribution distribution was least-squares was least-squares fitted using fitted a single-exponentialusing a single-exponential function, andfunction, the vertical and the broken vertical line lifetime distribution was least-squares fitted using a single-exponential function, and the vertical brokenillustrates line theillustrates value of the the va timelue of constant. the time constant. broken line illustrates the value of the time constant.

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2.7. Lack of BIS Effect on Single-Channel Conductance of KM Channels 2.7. Lack of BIS Effect on Single-Channel Conductance of KM Channels We next measured the amplitude and open probability of single KM channels at a series of voltages We next measured the amplitude and open probability of single KM channels at a series of ranging between −40 and +30 mV. Throughout the voltage range examined, averaged I-V relationships voltages ranging between -40 and +30 mV. Throughout the voltage range examined, averaged I-V of KM channels with or without BIS (1 µM) addition were analyzed and then compared (Figure7A). relationships of KM channels with or without BIS (1 μM) addition were analyzed and then compared In the control, fitting these single-channel amplitudes with a linear regression revealed K channels of (Figure 7A). In the control, fitting these single-channel amplitudes with a linear regressionM revealed 19.8 ± 0.4 pS (n = 9). The value for these channels did not differ significantly from that (19.7 ± 0.4 pS, KM channels of 19.8 ± 0.4 pS (n = 9). The value for these channels did not differ significantly from that n = 9, p > 0.05) during exposure to 1-µM BIS. It is clear from the results that addition of this compound (19.7 ± 0.4 pS, n = 9, p > 0.05) during exposure to 1-μM BIS. It is clear from the results that addition of is unable to modify single-channel conductance, although it can significantly exert a depressant on the this compound is unable to modify single-channel conductance, although it can significantly exert a probability of KM channels that would be open. depressant on the probability of KM channels that would be open.

2.4 1.0 2.0 0.8 1.6 0.6

pA 1.2 0.4 0.8 0.2

0.4 Relative open probability 0.0 0.0 -80 -60 -40 -20 0 20 40 -60 -40 -20 0 20 40 Δ Δ voltage (mV) voltage (mV) A B

FigureFigure 7.7.Effect Effect of of BIS BIS on I-Von I-Vrelationship relationship and activationand activation curve ofcurve KM channels of KM channels in GH3 cells. in GH (A)3 Averagedcells. (A) I-VAveragedrelationships I-V relationships of single KM of-channel single K currentsM-channel in currents the absence in the ( )absence and presence (■) and ( presence) of 1-µM (○ BIS) of (mean 1-μM ±BISSEM; (meann = ± 9 SEM; for each n = 9 data for each point). data The point). broken The line broken is pointed line is toward pointed the toward value ofthe# the value reversal of the potential reversal withpotential -70 mV. with (B -70) Activation mV. (B) Activation curves of KcurvesM channels of KM channels in the absence in the (absence) and presence(■) and presence () of 1- µ(□M) of BIS 1- (meanμM BIS± (meanSEM; n± =SEM; 9 for n each = 9 for data each point). data Channel point). Channel activity measuredactivity measured under cell-attached under cell-attached current recordingscurrent recordings was measured was measured at different at different potentials pote relativentials torelative the bath. to the The bath. smooth The curves smooth overlaid curves ontooverlaid each onto data each set were data least-squaresset were least-squares fitted with fi thetted Boltzmann with the Boltzmann equation (seeequation Materials (see Materials and Methods and forMethods details). for details).

2.8. Activation Curve of KM Channels Modified by the Presence of BIS 2.8. Activation Curve of KM Channels Modified by the Presence of BIS Figure7B shows the activation curve of K M channels taken with or without addition of 1-µM BIS. Figure 7B shows the activation curve of KM channels taken with or without addition of 1-μM BIS. In these experiments, channel open probabilities were measured at different voltages relative to the In these experiments, channel open probabilities were measured at different voltages relative to the bath. The plot of channel open probability of KM channels as a function of ∆voltage was derived and bath. The plot of channel open probability of KM channels as a function of Δvoltage was derived and then least-squares fitted with the Boltzmann function as described in Materials and Methods for details. then least-squares fitted with the Boltzmann function as described in Materials and Methods for In control (i.e., in the absence of BIS), Pmax = 0.99 ± 0.01, V1/2 = −19.2 ± 1.2 mV, q = 2.83 ± 0.05 e details. In control (i.e., in the absence of BIS), Pmax = 0.99 ± 0.01, V1/2 = −19.2 ± 1.2 mV, q = 2.83 ± 0.05 e (n = 9), whereas in the presence of 1-µM BIS, Pmax = 0.45 ± 0.01, V1/2 = −12.1 ± 0.9 mV, q = 2.85 ± 0.05 (n = 9), whereas in the presence of 1-μM BIS, Pmax = 0.45 ± 0.01, V1/2 = −12.1 ± 0.9 mV, q = 2.85 ± 0.05 e e (n = 9). Therefore, it is clear from these results that the presence of BIS not only produced a decrease (n = 9). Therefore, it is clear from these results that the presence of BIS not only produced a decrease in the maximal open probability of these channels, but also significantly shifted the activation curve in the maximal open probability of these channels, but also significantly shifted the activation curve along the voltage axis to depolarized potential by approximately 7 mV. In contrast, minimal change along the voltage axis to depolarized potential by approximately 7 mV. In contrast, minimal change on the gating charge of such activation curve was demonstrated in the presence of BIS. Consequently, on the gating charge of such activation curve was demonstrated in the presence of BIS. Consequently, as cells were exposed to 1-µM BIS, the difference in free energy (∆G = -q × F × V1/2, where F = 23,063 as cells− were1 −exposed1 to 1-μM BIS, the difference in free energy (ΔG = -q × F × V1/2, where F = 23,063 cal × V × e ) required for activation of KM channels was significantly reduced from 1.25 ± 0.04 to cal × V−1 × e−1) required for activation of KM channels was significantly reduced from 1.25 ± 0.04 to 0.79 0.79 ± 0.04 Kcal (n = 9, p < 0.05). ± 0.04 Kcal (n = 9, p < 0.05). + 2.9. Effect of BIS on Erg-Mediated K Current (IK(erg)) in GH3 Cells 2.9. Effect of BIS on Erg-Mediated K+ Current (IK(erg)) in GH3 Cells Previous studies have reported the importance of IK(erg) functionally expressed in pituitary cells Previous studies have reported the importance of IK(erg) functionally expressed in pituitary cells including GH3 cells [41,42]. Some of β-adrenoceptor blockers have been previously demonstrated including GH3 cells [41,42]. Some of β-adrenoceptor blockers have been previously demonstrated to suppress HERG K+ or other types of voltage-gated K+ channels [13,14]. As such, we further studied the possible perturbation by BIS on this type of K+ currents (i.e., IK(erg)). Interestingly, as cells were

Int. J. Mol. Sci. 2019, 20, 657 9 of 20 to suppress HERG K+ or other types of voltage-gated K+ channels [13,14]. As such, we further + studied the possible perturbation by BIS on this type of K currents (i.e., IK(erg)). Interestingly, as cellsInt. wereJ. Mol. Sci. exposed 2019, 20, tox FOR different PEER REVIEW concentrations of BIS, the amplitude of IK(erg) elicited by membrane 9 of 20 hyperpolarization progressively decreased in a concentration-dependent manner (Figure8A,B). exposed to different concentrations of BIS, the amplitude of IK(erg) elicited by membrane From nonlinear least-squares fit to the data points, the IC50 value needed to exert its inhibitory effect on hyperpolarization progressively decreased in a concentration-dependent manner (Figure 8A and 8B). the amplitude of deactivating IK(erg) was calculated to be 6.42 µM with a Hill coefficient of 1.2, and the From nonlinear least-squares fit to the data points, the IC50 value needed to exert its inhibitory effect agent at a concentration of 100 µM could nearly abolish current amplitude. Moreover, during continued on the amplitude of deactivating IK(erg) was calculated to be 6.42 μM with a Hill coefficient of 1.2, and presencethe agent of 3-µ atM a BIS, concentration subsequent of addition100 μM could of 10- nearlyµM PD-118057 abolish current significantly amplitude. attenuated Moreover, the during decrease of IK(erg)continuedproduced presence by this of agent. 3-μM PD-118057BIS, subsequent was previouslyaddition of 10- reportedμM PD-118057 to activate significantlyIK(erg) in attenuated heart cells [43]. Therefore,the decrease BIS could of IK(erg) effectively produced suppress by this agent.IK(erg) PD-118057amplitude, was despitepreviously its reported inhibitory to effectactivate on IK(erg)IK(erg) in to a lesserheart extent cells as [43]. compared Therefore, with BIS thatcould on effectivelyIK(M) described suppress above.IK(erg) amplitude, Similar todespite previous its inhibitory observations effect [14] showingon IK(erg) the abilityto a lesser of propranolol extent as compared and its derivative with that toon blockIK(M) described HERG channels, above. Similar BIS-induced to previous inhibition observations [14] showing the ability of propranolol and its derivative to block HERG channels, BIS- of IK(erg) tends to be independent of its agonistic effect on β-adrenergic receptors. induced inhibition of IK(erg) tends to be independent of its agonistic effect on β-adrenergic receptors. A 0 B

mV -50

-100 100 0 1000 2000 msec 80

0 60

40 -100

pA 20

c Percentage inhibition (%) 0 -200 b a 0.1 1 10 100 [BIS], μM -300 010002000 msec

+ FigureFigure 8. Inhibitory 8. Inhibitory effect effect of BISof BIS on onerg erg-mediated-mediated K K+ currents ( (IIK(erg)K(erg)) in) GH in GH3 cells.3 cells. In these In these experiments, experiments, + 2+ + we bathedwe bathed the cellsthe cells in high-K in high-K, Ca+, Ca2+-free-free solutionsolution and and the the pipette pipette used used was was filled filled with with K+-containing K -containing solution.solution. (A )(A Original) Original current current traces traces inin responseresponse to membrane membrane hyperpolarization hyperpolarization from from −10 −mV10 mV

(indicated(indicated in the in upperthe upper part). part). a: a: control; control; b: b: 1- 1-µμMM BIS;BIS; c: 3- μµMM BIS. BIS. (B (B) )Percentage Percentage inhibition inhibition of IK(erg) of I K(erg) producedproduced by different by different concentrations concentrations (0.3–100 (0.3–100µM) μM) of of BIS. BIS. Each Each cell cell was was hyperpolarized hyperpolarized from from −−1010 to to -80 mV, and-80 mV, current and current amplitudes amplitudes at the at end the of end hyperpolarizing of hyperpolarizing step step with with or or without without BIS BIS werewere measured and comparedand compared (mean ± (meanSEM; ± SEM;n = 8 n for = 8 eachfor each data data point). point). Smooth Smooth line represents represents the the best best fit to fit a tomodified a modified Hill function (see Materials and Methods for details). (C) Summary bar graph showing the effect of Hill function (see Materials and Methods for details). (C) Summary bar graph showing the effect of BIS and BIS plus PD-118057 on IK(erg) amplitude (mean ± SEM; n = 9 for each bar). IK(erg) elicited by BIS and BIS plus PD-118057 on I amplitude (mean ± SEM; n = 9 for each bar). I elicited by hyperpolarization from −10 to −K(erg)80 mV was measured at the beginning of each hyperpolarizingK(erg) pulse. − − hyperpolarizationPD: PD-118057 from(10 μM).10 * toSignificantly80 mV was different measured from control at the beginning(p < 0.05) and of each** significantly hyperpolarizing different pulse. PD: PD-118057from 3-μM BIS (10 aloneµM). group * Significantly (p < 0.05). different from control (p < 0.05) and ** significantly different from 3-µM BIS alone group (p < 0.05).

2.10. The Inhibition of Delayed-Rectifier K+ Current (IK(DR)) in GH3 cells Caused by the Presence of BIS

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Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW + 10 of 20 2.10. The Inhibition of Delayed-Rectifier K Current (IK(DR)) in GH3 cells Caused by the Presence of BIS

AnAn earlier earlier report report showed showed the the ability ability of ofcarvedilol carvedilol to suppress to suppress IK(DR)IK(DR) in NG108-15in NG108-15 neuronal neuronal cells [13].cells We [13 ].also We explored also explored whether whether or not or the not presence the presence of BIS of BISproduced produced any anyeffects effects on another on another types types of + 2+ Kof+ Kcurrentscurrents (e.g., (e.g., IK(DR)IK(DR)) in )these in these cells. cells. In Inthese these experiments, experiments, we we bathed bathed cells cells in in Ca Ca2+-free-free Tyrode’s Tyrode’s solution,solution, and, and, once once whole-cell whole-cell mode mode was was firmly firmly established, established, the the examined examined cells cells were were maintained maintained at −at50− mV50 mVand and a series a series of ofdepolarizi depolarizingng voltage voltage steps steps ranging ranging between between −−5050 and and +60 +60 mV mV was was then then delivered.delivered. BIS BIS at at a a concentration concentration of of 10 μµM was not foundfound toto havehave significantsignificant effecteffect ononI IK(DR)K(DR) elicitedelicited throughoutthroughout the the entire entire voltage-clamp voltage-clamp steps steps applied applied (Figure (Figure 9).9 ).However, However, upon upon cell cell exposure exposure to BIS to BIS(30 μ(30M),µ M),the theIK(DR)IK(DR) amplitudeamplitude waswas significantly significantly decreased. decreased. For For example, example, at atthe the level level of of +50 +50 mV, mV, current current amplitudeamplitude decreased decreased from 352 ±± 3131 to 235 ± 2525 pA pA (n (n == 11, 11, pp < 0.05). 0.05). Therefore, Therefore, distinct from IIK(M)K(M) oror IIK(erg)K(erg), the, the IK(DR)IK(DR) is relativelyis relatively resistant resistant to suppression to suppression by byBIS. BIS.

A +60 mV B

-50 mV 400

control 300

200 pA

100

BIS 0

-60 -40 -20 0 20 40 60 mV 100 pA 300 msec

+ Figure 9. Effect of BIS on delayed-rectifier K current (IK(DR)) in GH3 cells. In these whole-cell current 2+ Figurerecordings, 9. Effect cells of were BIS bathedon delayed-rectifier in Ca -free Tyrode’sK+ current solution. (IK(DR)) Thein GH examined3 cells. In cells these were whole-cell held at −current50 mV recordings,and a series cells of voltagewere bathed step in ranging Ca2+-free between Tyrode’s−50 solution. and +60 The mV. examined (A) Superimposed cells were held current at −50 traces mV andobtained a series in theof absencevoltage (upper)step ranging and presence between (lower) −50 and of 30-+60µ MmV. BIS. (A The) Superimposed uppermost part current indicates traces the obtainedvoltage protocol in the absence used and (upper) arrowhead and presence is the zero (lower) current of 30- level.μM ( BBIS.) Averaged The uppermostI-V relationships part indicates of IK(DR) the voltageobtained protocol in the absenceused and ( arrowhea) and presenced is the ( zero) of 30-currentµM BIS level. (mean (B) Averaged± SEM; n I-V= 11 relationships for each data of point). IK(DR) obtainedCurrent amplitudein the absence was (■ measured) and presence at the ( end○#) of of 30- eachμM depolarizingBIS (mean ± SEM; step fromn = 11 a for holding each data potential point). of Current−50 mV. amplitude was measured at the end of each depolarizing step from a holding potential of −50 mV. 2.11. Effect of BIS on Spontaneous Action Potentials (APs) in GH3 Cells

2.11. EffectIn a separate of BIS on set Spontaneou of experiments,s Action we Potentials explored (APs) whether in GH the3 Cells presence of BIS perturbs any changes in membrane potential recorded from these cells. As shown in Figure 10A, as the current-clamp In a separate set of experiments, we explored whether the presence of BIS perturbs any changes voltage recordings were established, spontaneous APs with a firing frequency of 0.85 ± 0.06 Hz (n = 8) in membrane potential recorded from these cells. As shown in Figure 10A, as the current-clamp were readily observed. As cells were exposed to BIS, membrane became depolarized and the firing voltage recordings were established, spontaneous APs with a firing frequency of 0.85 ± 0.06 Hz (n = frequency was concomitantly raised, as evidenced by a significant raise in the firing frequency to 8) were readily observed. As cells were exposed to BIS, membrane became depolarized and the firing 1.62 ± 0.06 Hz (n = 8, p < 0.05) during the exposure to 3-µM BIS. In continued presence of 3-µM BIS, frequency was concomitantly raised, as evidenced by a significant raise in the firing frequency to 1.62 subsequent addition of 10 µM flupirtine significantly attenuated BIS-induced increase in the firing of ± 0.06 Hz (n = 8, p < 0.05) during the exposure to 3-μM BIS. In continued presence of 3-μM BIS, spontaneous APs to 1.16 ± 0.06 Hz (n = 8, p < 0.05); however, that of 10 µM ractopamine did not have subsequent addition of 10 μM flupirtine significantly attenuated BIS-induced increase in the firing of any effects on it (Figure 10B). It is therefore conceivable that the observed effect of BIS on the firing spontaneous APs to 1.16 ± .0.06 Hz (n = 8, p < 0.05); however, that of 10 μM ractopamine did not have patterns of GH cells appears to be independent of binding to β-adrenergic receptors and could be any effects on it3 (Figure 10B). It is therefore conceivable that the observed effect of BIS on the firing partly explained by its suppression of IK(M). patterns of GH3 cells appears to be independent of binding to β-adrenergic receptors and could be partly explained by its suppression of IK(M).

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A 0 control -20

-40 mV -60

-80 0246810 sec 0 BIS -20

-40 mV -60

-80 0246810 sec Figure 10. Effects of BIS on spontaneous action potentials (APs) in GH3 cells. In these current-clamp voltage recordings, cells were bathed in normal Tyrode’s solution containing 1.8 mM CaCl2, and the recordingFigure 10. pipette Effects was of filledBIS on with spontaneous K+-containing action solution. potentials (A) (APs) Original in GH potential3 cells. traceIn these obtained current-clamp in the absencevoltage (upper) recordings, and presencecells were (lower) bathed of in 3- normµM BIS.al Tyrode’s (B) Summary solution bar containing graph showing 1.8 mM the CaCl effects2, and of the recording pipette was filled with K+-containing solution. (A) Original potential trace obtained in the BIS, BIS plus ractopamine, and BIS plus flupirtine on the firing frequency of spontaneous APs in GH3 cellsabsence (mean (upper)± SEM; andn = presence 8 for each (lower) bar). a:of control;3-μM BIS. b: ( 1-Bµ) SummaryM BIS c: 3- barµM graph BIS; d: showing 3-µM BIS the plus effects 10 ofµM BIS, ractopamine;BIS plus ractopamine, e: 3-µM BIS and plus BIS 10 plusµM flupirtine flupirtine. on * Significantlythe firing frequency different of fromspontaneous control (APsp < 0.05)in GH and3 cells ** significantly(mean ± SEM; different n = 8 fromfor each the 3-bar).µM BISa: control; alone group b: 1-μ (pM< BIS 0.05). c: 3-μM BIS; d: 3-μM BIS plus 10 μM ractopamine; e: 3-μM BIS plus 10 μM flupirtine. * Significantly different from control (p < 0.05) and ** 2.12. Suppressivesignificantly Effect different of BIS from onI theK(M) 3-inμM Pituitary BIS alone R1220 group Cells (p < 0.05).

We next wanted to test if IK(M) in another type of pituitary cells (e.g., pituitary R1220 cells) can be2.12. influenced Suppressive by BIS. Effect This of BIS type on ofIK(M) rat in pituitary Pituitary cells,R1220 originally Cells derived from ScienCell (Carlsbad,

CA, USA),We next was previouslywanted to test reported if IK(M) toin expressanotherβ type1-adrenergic of pituitary receptors cells (e.g., [8]. pituitary As depicted R1220 in Figurecells) can 11 ,be asinfluenced cells were exposed by BIS. toThis different type of BIS rat concentrations, pituitary cells, the originally amplitude derived of IK(M) fromin response ScienCell to (Carlsbad, depolarizing CA), stepwas was previously progressively reported decreased, to express in β combination1-adrenergic withreceptors a slowing [8]. As in depic activationted in Figure time course 11, as ofcellsIK(M) were. Moreover,exposed withto different the presence BIS conc of 1-entrations,µM BIS, further the amplitude addition of of ML-213 IK(M) in (10responseµM), an to activator depolarizing of IK(M) step[44 was], wasprogressively able to attenuate decreased, BIS-induced in combination inhibition with of IK(M) a slowing, despite in inability activation of further time course isoproterenol of IK(M). additionMoreover, towith exert the any presence effect on of such 1-μ block.M BIS, These further results addition thus appearof ML-213 to be (10 indistinguishable μM), an activator from of I thoseK(M) [44], described was able aboveto attenuate in GH3 cells.BIS-induced inhibition of IK(M), despite inability of further isoproterenol addition to exert any effect on such block. These results thus appear to be indistinguishable from those described 2.13. Effect of BIS on I Recorded from Hippocampal mHippoE-14 Cells above in GH3 cells.K(M)

In a final set of experiments, we wanted to determine whether or not IK(M) inherently in central neurons (e.g., hippocampal mHippoE-14 cells) [21] is subject to suppression by this compound. The experimental profiles used were similar to those for pituitary cells. As depicted in Figure 12A,B, the IK(M) elicited by different depolarizing steps was suppressed by the presence of BIS. For example, at the level of −10 mV, the exposure to 3-µM BIS caused a significant reduction of IK(M) amplitude from 109 ± 9 pA to 27 ± 6 pA (n = 9, p < 0.05). After washout of BIS, current amplitude returned to 98 ± 9 pA (n = 9, p < 0.05). The averaged I-V relationships of IK(M) obtained with or without BIS addition are illustrated in Figure 12B. However, isoproterenol (1 µM) had little or no effects on IK(M) amplitude (108 ± 9 pA [control] versus 107 ± 10 pA [in the presence of 1 µM isoproterenol], n = 9, p > 0.05). Therefore, the magnitude of BIS-induced block of IK(M) taken from mHippoE-14 cells is indistinguishable from those described above in pituitary cells.

Int. J. Mol. Sci. 2019, 20, 657 12 of 20 Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 12 of 20

A 0 B

mV -40 300 0 500 1000 1500 2000 msec *,** * 0 200

pA * b c -100 a

100

pA -200

-300 0 (a) (b) (c) (d)

-400 0 500 1000 1500 2000 msec

Figure 11.FigureInhibitory 11. Inhibitory effectof effect BIS of on BISIK(M) on IrecordedK(M) recorded from from rat rat pituitary pituitary R1220 R1220 cells.cells. In In this this set set of of recordings,recordings, similar similar experimental experimental conditions conditions to to that that in in GH GH3 3 cellscells described above above were were applied. applied. (A) (A) OriginalOriginalIK(M) elicitedIK(M) elicited by membrane by membrane depolarization depolarization from from− −5050 to −1010 mV. mV. a:a: control; control; b: 0.3- b: 0.3-μMµ BISM BISand c: 1 μM BIS. (B) Summary bar graph showing effect of BIS and BIS plus flupirtine on IK(M) in these cells and c: 1 µM BIS. (B) Summary bar graph showing effect of BIS and BIS plus flupirtine on IK(M) in these cells (mean(mean± SEM; ± SEM;n = 12n = for12 for each each bar). bar). a: a: control; control;b: b: 0.3-0.3-µμM BIS; c: c: 1- 1-μµMM BIS; BIS; d: d:1-μ 1-Mµ MBIS BIS plus plus 10-μ 10-M µML-M 213. * Significantly different from control (p < 0.05) and ** significantly different from 1-μM BIS alone ML-213. * Significantly different from control (p < 0.05) and ** significantly different from 1-µM BIS Int. J. Mol. Sci.group 2019, (p20 ,< x 0.05). FOR PEER REVIEW 13 of 20 alone group (p < 0.05).

2.13. Effect of BIS on IK(M) Recorded from Hippocampal mHippoE-14 Cells A -10 mV In a final set of experiments, we wanted to determineB whether or not IK(M) inherently in central neurons (e.g., hippocampal mHippoE-14-50 mV cells) [21] is subject to suppression by this compound. The experimental profiles used were similar to those for pituitary cells. As depicted in Figure 12A and 0 12B, the IK(M) elicited by different depolarizing steps was suppressed by the presence of BIS. For control example, at the level of −10 mV, the exposure to-20 3-μM BIS caused a significant reduction of IK(M) amplitude from 109 ± 9 pA to 27 ± 6 pA (n = 9, p < 0.05). After washout of BIS, current amplitude returned to 98 ± 9 pA (n = 9, p < 0.05). The averaged-40 I-V relationships of IK(M) obtained with or without BIS addition are illustrated in Figure 12B. However, isoproterenol (1 μM) had little or no effects on -60 IK(M) amplitude (108 ± 9 pA [control] versus 107 ± 10 pA [in the presence of 1 μM isoproterenol], n = 9, pA p > 0.05). Therefore, the magnitude of BIS-induced-80 block of IK(M) taken from mHippoE-14 cells is indistinguishable from those described above in pituitary cells. BIS -100

-120

-50 -40 -30 -20 -10 40 pA mV 300 msec

Figure 12. Effect of BIS on IK(M) in hippocampal mHippoE-14 cells. In these experiments, we bathed Figure 12. Effect of BIS on IK(M) in hippocampal mHippoE-14 cells. In these experiments, we bathed the cells in high-K+, Ca2+-free solution, and the examined cell was depolarized from −50 mV to various the cells in high-K+, Ca2+-free solution, and the examined cell was depolarized from −50 mV to various voltages ranging between −50 and −10 mV. (A) Original current traces obtained in the absence (upper) voltages ranging between −50 and −10 mV. (A) Original current traces obtained in the absence (upper) µ and presenceand presence (lower) (lower) of 3of 3M μM BIS. BIS. The The uppermost uppermost partpart indicate indicatess the the voltage voltage protocol protocol applied applied and and ± arrowheadarrowhead is the is zero the zero current current level. level. (B )(B Averaged) AveragedI-V I-Vrelationship relationship ofof IK(M) (mean(mean ± SEM;SEM; n = 9n for=9 each for each data point).data point).: control; ■: control;: □ 3: µ3 MμM BIS. BIS. Current Current amplitudeamplitude was was measured measured at atthe the end end of each of each voltage voltage step. step.

3. Discussion

BIS-induced suppression of IK(M) presented herein was found to be attenuated by a further application of flupirtine, but not by isoproterenol or ractopamine, the agonist of β-adrenergic receptor. The present results are of particular importance, as they suggest that the inhibition of both IK(M) and IK(erg) caused by BIS is direct and appears to be unnecessarily linked to its binding to β- adrenergic receptor, although binding to β1-adrenergic receptor was previously reported to influence pituitary function [7,8,11,12,45]. However, distinguishable from the inhibitory effect of carvedilol, a non-selective blocker of β-adrenergic receptors, on the amplitude and inactivation kinetics IK(DR) [13], BIS at a concentration greater than 10 μM slightly suppressed IK(DR) amplitude with no discernible change in the inactivation time course of the current. A previous report has demonstrated the ability of BIS to reverse the down-regulation in mRNA expression of small-conductance Ca2+-activated K+ channels [15]. In our study, neither continued presence of BIS, further addition of neither diazoxide, GMQ, nor 9-phenanthrol produced attenuating effects on its suppression of IK(M). Diazoxide and 9-phenanthrol are known to stimulate ATP-sensitive and intermediate-conductance Ca2+-activated K+ channels, respectively, while 2-guanidine-4- methylquinazoline (GMQ) was reported to enhance the activity of large-conductance Ca2+-activated K+ channels [39,40]. In this scenario, the observed effect of BIS on whole-cell IK(M) inherently in pituitary GH3 cells virtually is not involved in the suppression of either ATP-sensitive or Ca2+- activated K+ channels. The inhibitory effect on IK(M) by BIS could be predominantly responsible for its decrease of macroscopic IK(M) amplitude carried through KM channels. Block of IK(M) caused by BIS is not instantaneous, but develops with time after the channels are opened upon rapid membrane depolarization, producing an apparent slowing in current activation. Deactivating current of IK(M) in the presence of BIS showed a blunted peak and an increased decay that is consistent with the possibility that the closing (i.e., deactivating process) of channels was raised

Int. J. Mol. Sci. 2019, 20, 657 13 of 20

3. Discussion

BIS-induced suppression of IK(M) presented herein was found to be attenuated by a further application of flupirtine, but not by isoproterenol or ractopamine, the agonist of β-adrenergic receptor. The present results are of particular importance, as they suggest that the inhibition of both IK(M) and IK(erg) caused by BIS is direct and appears to be unnecessarily linked to its binding to β-adrenergic receptor, although binding to β1-adrenergic receptor was previously reported to influence pituitary function [7,8,11,12,45]. However, distinguishable from the inhibitory effect of carvedilol, a non-selective blocker of β-adrenergic receptors, on the amplitude and inactivation kinetics IK(DR) [13], BIS at a concentration greater than 10 µM slightly suppressed IK(DR) amplitude with no discernible change in the inactivation time course of the current. A previous report has demonstrated the ability of BIS to reverse the down-regulation in mRNA expression of small-conductance Ca2+-activated K+ channels [15]. In our study, neither continued presence of BIS, further addition of neither diazoxide, GMQ, nor 9-phenanthrol produced attenuating effects on its suppression of IK(M). Diazoxide and 9-phenanthrol are known to stimulate ATP-sensitive and intermediate-conductance Ca2+-activated K+ channels, respectively, while 2-guanidine-4-methylquinazoline (GMQ) was reported to enhance the activity of large-conductance Ca2+-activated K+ channels [39,40]. In this scenario, the observed effect of BIS on whole-cell IK(M) inherently in pituitary GH3 cells virtually is not involved in the suppression of either ATP-sensitive or Ca2+-activated K+ channels. The inhibitory effect on IK(M) by BIS could be predominantly responsible for its decrease of macroscopic IK(M) amplitude carried through KM channels. Block of IK(M) caused by BIS is not instantaneous, but develops with time after the channels are opened upon rapid membrane depolarization, producing an apparent slowing in current activation. Deactivating current of IK(M) in the presence of BIS showed a blunted peak and an increased decay that is consistent with the possibility that the closing (i.e., deactivating process) of channels was raised by unbinding of the BIS molecule. Moreover, as the falling phase upon return to hyperpolarizing potential (i.e., the down sloping ramp) was prolonged, the IK(M) amplitudes were decreased exponentially. The magnitude of BIS-induced block on such deactivating currents was notably increased. The blocking site of this agent thus appears to be located within the channel pore only when the channel is open. KCNQ2/3 mRNA expression was detected in GH3 and mHippoE-14 cells; however, the present finding showed a lack of effect of BIS on single-channel conductance of KM channels in GH3 cells, suggesting that the interaction of the channel with it seems to be secondary to alterations that are remote from the pore region of the channel. Clinically relevant serum concentration of BIS was recently reported to range between 3.8 and 71.1 ng/mL (or 0.011 and 0.22 µM) [46]. These values are similar to the IC50 required for its suppression of IK(M) presented herein. In this study, the IC50 value required for BIS-induced block of IK(M) or IK(erg) inherently in GH3 cells was 1.21 and 6.42 µM, respectively. This agent tends to be selective for IK(M) over IK(erg) or IK(DR). There was also a depolarizing shift in the activation curve of KM channels during exposure to BIS, strongly suggesting that this compound could alter the voltage dependence of channel activation accompanied by a decrease of free energy for KM-channel activation. During high frequency firing, the amplitude of IK(M) was reduced and the sensitivity to BIS was attenuated. As the duration of the falling phase upon return from membrane potential of −10 to −50 mV increased, the magnitude of BIS-induced block of IK(M) became raised. Taken together, the sensitivity of neurons or pituitary cells to BIS could depend not only on the BIS concentration achieved, but also on the pre-existing level of the resting potential, the discharge pattern or frequency, or their combinations. Our study showed that in pituitary cells, the presence of BIS does not appear to bind to β-adrenergic receptors exclusively, although the activity of these receptors might be constitutively active [47]. This compound was capable of suppressing IK(M) and IK(erg) directly and effectively. Under current-clamp recordings, BIS was effective at depolarizing the cell and raising the firing rate of spontaneous APs in GH3 cells. Subsequent addition of flupirtine, yet not ractopamine, could attenuate BIS-enhanced firing. Therefore, apart from its antagonistic effect on β-adrenergic receptors, Int. J. Mol. Sci. 2019, 20, 657 14 of 20 the inhibition by BIS of these K+ currents may also conceivably contribute to its effect on the cellular function (e.g., stimulus-secretion coupling) in these cells. However, it also needs to be noted that the results presented here were derived from cell lines. Whether similar findings occurring in native cells still remains to be clarified. Neither eugenolol nor eugenodilol was found to suppress IK(M) in GH3 cells, although these newly designed compounds possess blocking actions on β-adrenergic receptors [48,49]. BIS or its structurally similar compounds may thus be a valuable tool for probing the structure and function of KM or Kerg channels, because the pore region of the channel protein to which they bind is of particular relevance for an open-channel blockade. In addition to its antagonistic effect on β1-adrenergic receptors, the blockade of KM and Kerg channels by BIS, together with an increase in the firing of spontaneous APs, may be responsible for its effects on pituitary or neuronal excitability. To what extent BIS-induced suppression of these K+ currents is responsible for its additional effectiveness with favorable metabolic profiles [2,50] has yet to be further delineated. Caution needs to be made in attributing its use to the selective blocking of β1-adrenergic receptors.

4. Materials and Methods

4.1. Chemicals and Solutions (±)-Bisoprolol hemifumarate (BIS, zebeta®, (E)-but-2-enedioic acid;1-(propan-2-ylamino)- 1 3-[4-(2-propan-2-yloxyethoxymethyl)phenoxy]propan-2-ol, C13H31NO4· 2 C4H4O4), 2-guanidine- 4-methylquinazoline (GMQ), ML-213, PD-118057, and 9-phenanthrol were obtained from Tocris (Bristol, UK), while carvedilol, diazoxide, ethidium bromide, flupirtine, isoproterenol, ivabradine, linopirdine, ractopamine and tolbutamide were from Sigma-Aldrich (St. Louis, MO, USA). Pioglitazone was obtained from Takeda Pharmaceutical (Osaka, Japan), and parecoxib was from Pfizer Inc. (New York, NY, USA). Eugenolol and eugenodilol were gifts from Dr. Jwu-Lai Yeh, Department of Pharmacology, Kaohsiung Medical University, Kaohsiung City, Taiwan. Unless stated otherwise, the cell culture media, L-glutamine, horse serum and fetal calf serum were obtained from Invitrogen (Carlsbad, CA, USA). All other chemicals were commercially available and of analytical reagent grade. The composition of HEPES-buffered normal Tyrode’s solution was as follows (in mM): NaCl 136.5, KCl 5.4, CaCl2 1.8, MgCl2 0.53, glucose 5.5, and HEPES-NaOH buffer 5.5 (pH 7.4). To record membrane potential or IK(M), the recording pipettes were filled with the solution (in mM); K-aspartate 130, KCl 20, KH2PO4 1, MgCl2 1, EGTA 0.1, Na2ATP 3, Na2GTP 0.1, and HEPES-KOH buffer 5 (pH 7.2). + To record whole-cell IK(M), high K -bathing solution was composed of the following (in mM): KCl 145, MgCl2 0.53, and HEPES-KOH 5 (pH 7.4). To measure the activity of single KM channels, the pipette solution was composed of the following (in mM): NaCl 136.5, KCl 5.4, MgCl2 0.53, and HEPES-NaOH buffer 5 (pH 7.4). All solutions were prepared using deionized water from a Milli-Q water purification system (APS Water Services, Inc., Van Nuys, CA, USA). The pipette solution and culture medium were commonly filtered on the day of use with Acrodisc® syringe filter with 0.2 µm of Supor® membrane (Pall Corp., Port Washington, NY, USA).

4.2. Cell Preparations

GH3 pituitary tumor cells, obtained from the Bioresources Collection and Research Center ([BCRC-60015]; Hsinchu, Taiwan; originally derived from ATCC [CCL-82.1TM], were maintained in Ham’s F-12 medium supplemented with 15% horse serum (v/v), 2.5% fetal calf serum (v/v), and 2 mM L-glutamine in a humidified environment of 5% CO2/95% air. Rat pituitary cells (#R1220) were purchased from ScienCell Research Laboratories, Inc. (Carlsbad, CA, USA). These cells reported to express the receptors of gonadotropin releasing hormone (GnRH) were isolated from neonate day-8 CD® rats and cryopreserved in primary cultures with further purification and expansion (https://www.sciencellonline.com/products-services/primary-cells/animal/cell-types/ Int. J. Mol. Sci. 2019, 20, 657 15 of 20 pituitarycells/rat-pituitary-cells.html)[51,52]. Cells were routinely grown in Epithelial Cell Medium (Cat #4101; ScienCell). Embryonic mouse hippocampal cell line (mHippoE-14, CLU198) was obtained from Cedarlane CELLutions Biosystems, Inc. (Burlington, ON, Canada) [39,53,54]. Cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (v/v) and 2 mM L-glutamine. The culture medium was changed every two to three days, and cells underwent passaged when they reached confluence. The experiments were commonly performed 5 or 6 days after cells had been cultured (60–80% confluence).

4.3. RNA Isolation and Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) To detect the expression of rat KCNQ2 and KCNQ3 mRNAs in different types of cells including GH3, R1220 cells, a semiquantitative RT-PCR assay was performed. Total RNA samples were extracted from mHippoE-14 cells with TRIzol reagent (Invitrogen) and reverse-transcribed into complementary DNA using Superscript II reverse-transcriptase (Invitrogen). The sequences of forward and reverse oligonucleotide primers used for KCNQ2 were 50-CCCTGAAAGTCCAAGAGCAG-30 and 50-AGGCCCCATAGGTTTGAGTT-30, respectively, while those for KCNQ3 were 50- GTGGCTTCA GCATCTCACAA-30 and 50-CTTGTTGGAAGGGGTCCATA-30, respectively. Amplication of KCNQ2 or KCNQ3 was made using PCR SuperMix from Invitrogen under the following conditions: 35 cycles composed of 30 sec denaturation at 95 ◦C. 30 sec primer annealing at 62 ◦C, 1 min extension at 72 ◦C, and followed by 72 ◦C for the final extension for 2 min. PCR products were analyzed on 1.5% (v/v) agarose gel containing ethidium bromide and then visualized under ultraviolet light. Optical densities of DNA bands were scanned and quantified by AlphaImager 2200 (ProteinSimple; Santa Clara, CA, USA).

4.4. Electrophysiological Measurements Immediately prior to the experiment, cells were dissociated and an aliquot of cell suspension was transferred to a custom-made recording chamber mounted on the stage of a DM-IL inverted microscope (Leica, Wetzlar, Germany). There were immersed at room temperature (20–25 ◦C) in HEPES-buffered normal Tyrode’s solution, the composition of which is described above. The patch electrodes were fabricated from borosilicate glass capillaries (No. 34500; Kimble Products, Vineland, NJ, USA) on a Narishige PP-83 puller (Narishige, Tokyo, Japan) or a P-97 Flaming/Brown puller (Sutter, Novato, CA, USA), and electrode tips were fire-polished with MF-83 microforge (Narishige). Their resistances in standard pipette and bathing solutions ranged from 3 to 5 MΩ. Recordings of membrane potential or ion currents were measured in the whole-cell or cell-attached mode of the patch-clamp technique with an RK-400 patch-clamp amplifier (Bio-Logic, Claix, France) [42]. The liquid junction potentials were corrected shortly before seal formation was established.

4.5. Data Recordings The data comprising both potential and current traces were stored online in an Acer SPIN−5 touchscreen laptop computer (SP513-52N-55WE; Taipei, Taiwan) at 10 kHz equipped with Digidata 1440A interface (Molecular Devices, Inc., Sunnyvale, CA, USA), which was used for the analog-to-digital/digital-to-analog conversion. During the recordings, the latter device was controlled by pCLAMP 10.7 software (Molecular Devices) run under Windows 10 (Redmond, WA, USA), and the signals were simultaneously monitored on LCD monitor through a USB type-C connection. Current signals were low-pass filtered at 2 kHz with a FL-4 four-pole Bessel filter (Dagan, Minneapolis, MN, USA) to minimize background noise. Through digital-to-analog conversion, various pCLAMP-generated voltage-clamp profiles with either rectangular or ramp waveforms were applied to determine the current-voltage (I-V) relationship of IK(M), IK(erg) or IK(DR). As high-frequency stimuli were necessarily applied to the cell, an Astro-med Grass S88X dual output pulse stimulator (Grass Technologies, West Warwick, RI, USA) was used. After the data were digitally collected, we later Int. J. Mol. Sci. 2019, 20, 657 16 of 20 analyzed them using different analytical tools that include LabChart 7.0 program (AD Instruments; Gerin, Tainan, Taiwan), OriginPro 2016 (Microcal, Northampton, MA, USA), or custom-made macros built under Microsoft Excel® 2013 (Redmond).

4.6. Data Analyses

To determine percentage inhibition of BIS on IK(M) or IK(erg), we compared the current amplitudes + during cell exposure to different BIS concentrations. To record IK(M), we bathed the cells in high-K , 2+ Ca -free solution, and depolarizing step from −50 to −10 mV was applied, while, to measure IK(erg), the examined cell was hyperpolarized from −10 to −100 mV. The concentration-response data for inhibition of IK(M) or IK(erg) were least-squares fitted by a modified Hill function. That is,

E × [BIS]nH Percentage inhibiton = max , nH nH IC50 + [BIS] where [BIS] represents the BIS concentration; IC50 and nH are the concentration required for 50% inhibition and the Hill coefficient, respectively; and Emax is the maximal inhibition of IK(M) or IK(erg) induced by this drug.

+ 4.7. Analyses of Single M-Type K (KM) Channels

Single KM-channel currents recorded from GH3 cells or mHippoE-14 neurons were analyzed using pCLAMP 10.7. We determined single-channel amplitudes taken with or without the BIS addition by fitting Gaussian distributions to the amplitude histograms of the closed and open states. The probabilities of KM channel that would be open were defined as N·PO, which is estimated using the following expression: A1 + 2A2 + 3A3 + ... + nAn N·PO = A0 + A1 + A2 + ... + An where N is the number of active channels in the patch, A0 the area under the curve of an all points histogram corresponding to the closed state, and A1 ... An the histogram area that indicate the level of the distinct open state for 1 to n channels in the patch examined. The single-channel conductance of KM channels was calculated using a linear regression with averaged values of single-channel amplitudes measured at different levels of membrane potentials relative to the bath. Open lifetime distributions of KM channels taken with or without BIS addition were least-squares fitted with logarithmically scaled bin width. To determine voltage-dependence of the inhibitory effect of BIS on the activity of KM channels, the patch with or without BIS addition was held at different membrane potentials. The activation curve of KM-channel openings taken with or without addition of BIS (3 µM) was fitted by the Boltzmann equation: P = max Relative open probability −(V−V )qF , [ 1/2 ] 1 + e RT where Pmax is the maximal probability of KM-channel openings in the control (i.e., in the absence of BIS) maintained at +30 mV relative to the bath, V1/2 the voltage at which half-maximal activation of KM channels occurs, q the apparent gating charge, F Faraday’s constant, R the universal gas constant, T absolution temperature, and F/RT = 0.04 mV−1.

4.8. Statistical Analyses The linear or nonlinear (e.g., sigmoidal or exponential curves) least-squares fitting routines were appropriately estimated using either the Solver add-in bundled with Microsoft ExcelTM 2013 (Redmond) or OriginPro 2016 (OriginLab, Northampton, MA, USA). The values appearing in this study are provided as the mean values ± standard error of mean (SEM) with sample sizes (n) indicating the number of cells from which the experimental data were taken, and error bars are plotted as SEM. Int. J. Mol. Sci. 2019, 20, 657 17 of 20

In this study, we intended to make assertions about the variability of means that could be collected from a random cohort derived from the population concerned. For this reason, the standard error could be more appropriate than the standard deviation. The paired or unpaired Student’s t-test, or one-way analysis of variance followed by post-hoc Fisher’s least-significance difference test for multiple-group comparisons, were implemented for the statistical evaluation of differences among means. We used non-parametric Kruskal-Wallis test, as the assumption of normality underlying ANOVA was violated. Statistical analyses were performed using IBM SPSS® version 20.0 (IBM Corp., Armonk, NY, USA). A difference with a p value < 0.05 was considered statistically significant, unless otherwise stated.

Author Contributions: Conceptualization, E.C.S. and S.-N.W.; validation, E.C.S., N.-P.F.; formal analysis, S.-N.W.; investigation, S.-N.W.; resources, S.Y.K.; data curation, N.-P.F.; writing—original draft preparation, E.C.S.; writing—review and editing, N.-P.F.; visualization, E.C.S.; supervision, S.-N.W.; project administration, S.Y.K.; funding acquisition, S.-N.W. Funding: This study was supported in part by National Cheng Kung University (D106-35A13, D107-F2519 and NCKUH-10709001 to S.N. Wu), Tainan City, Taiwan, as well as in part by An Nan Hospital, China Medical University. (ANHRF107-13 to E.C.S.). Acknowledgments: The authors would like to express our appreciation to Kaissen Lee for helpful assistance in this work, and Kenny So for helping with the English language editing. S.N.W. received a Talent Award for the Outstanding Researchers from Ministry of Education, Taiwan. Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations

AP action potential BIS bisoprolol erg ether-à-go-go-related gene GMQ 2-guanidine-4-methylquinazoline I-V current versus voltage IC50 the concentration required for 50% inhibition IK(DR) delayed-rectifier K+ current IK(erg) erg-mediated K+ current IK(M) M-type K+ current Kerg erg-mediated K+ channel KM channel M-type K+ channel SEM standard error of mean τact activation time constant τdeact deactivation time constant.

References

1. Vasomotor dysfunction in POTS patients. Rev. Port. Cardiol. 2000, 19, 1163–1170. 2. Agabiti Rosei, E.; Rizzoni, D. Metabolic profile of nebivolol, a β-adrenoceptor antagonist with unique characteristics. Drugs 2007, 67, 1097–1107. [CrossRef][PubMed] 3. Nakamura, K.; Murakami, M.; Miura, D.; Yunoki, K.; Enko, K.; Tanaka, M.; Saito, Y.; Nishii, N.; Miyoshi, T.; Yoshida, M.; et al. Beta-Blockers and Oxidative Stress in Patients with Heart Failure. Pharmaceuticals 2011, 4, 1088–1100. [CrossRef][PubMed] 4. Zhang, C.; He, S.; Li, Y.; Li, F.; Liu, Z.; Liu, J.; Gong, J. Bisoprolol protects myocardium cells against ischemia/reperfusion injury by attenuating unfolded protein response in rats. Sci. Rep. 2017, 7, 11859. [CrossRef][PubMed] 5. Bulpitt, C.J.; Connor, M.; Schulte, M.; Fletcher, A.E. Bisoprolol and nifedipine retard in elderly hypertensive patients: Effect on quality of life. J. Hum. Hypertens. 2000, 14, 205–212. [CrossRef][PubMed] 6. Sigaroudi, A.; Kinzig, M.; Wahl, O.; Stelzer, C.; Schroeter, M.; Fuhr, U.; Holzgrabe, U.; Sorgel, F. Quantification of bisoprolol and metoprolol in simultaneous human serum and cerebrospinal fluid samples. Pharmacology 2018, 101, 29–34. [CrossRef][PubMed] Int. J. Mol. Sci. 2019, 20, 657 18 of 20

7. Soloviev, D.V.; Matarrese, M.; Moresco, R.M.; Todde, S.; Bonasera, T.A.; Sudati, F.; Simonelli, P.; Magni, F.; Colombo, D.; Carpinelli, A.; et al. Asymmetric synthesis and preliminary evaluation of (R)- and 11 (S)-[ C]bisoprolol, a putative β1-selective adrenoceptor radioligand. Neurochem. Int. 2001, 38, 169–180. [CrossRef] 8. Janssens, K.; Krylyshkina, O.; Hersmus, N.; Vankelecom, H.; Denef, C. β1-Adrenoceptor expression in rat anterior pituitary gonadotrophs and in mouse αT3-1 and LβT2 gonadotrophic cell lines. Endocrinology 2008, 149, 2313–2324. [CrossRef] 9. Ul Haq, R.; Liotta, A.; Kovacs, R.; Rosler, A.; Jarosch, M.J.; Heinemann, U.; Behrens, C.J. Adrenergic modulation of sharp wave-ripple activity in rat hippocampal slices. Hippocampus 2012, 22, 516–533. [CrossRef] 10. Ronzoni, G.; Del Arco, A.; Mora, F.; Segovia, G. Enhanced noradrenergic activity in the amygdala contributes to hyperarousal in an animal model of PTSD. Psychoneuroendocrinology 2016, 70, 1–9. [CrossRef] 11. Sun, L.; Wang, S.; Lin, X.; Tan, H.; Fu, Z. Early Life Exposure to Ractopamine Causes Endocrine-Disrupting Effects in Japanese Medaka (Oryzias latipes). Bull. Environ. Contam. Toxicol. 2016, 96, 150–155. [CrossRef] [PubMed] 12. Nurmamedova, G.S.; Gumbatov, N.B.; Mustafaev, I.I. Level of hormones of pituitary-gonadal axis, penile blood flow and sexual function in men with arterial hypertension during monotherapy with bisoprolol and nebivolol. Kardiologiia 2007, 47, 50–53. [PubMed] 13. Liu, C.P.; Chiang, H.T.; Jan, C.R.; Wu, S.N. Mechanism of carvedilol-induced block of delayed rectifier K+ current in the NG108-15 neuronal cell line. Drug Dev. Res. 2003, 58, 196–208. [CrossRef] 14. Dupuis, D.S.; Klaerke, D.A.; Olesen, S.P. Effect of β-adrenoceptor blockers on human ether-a-go-go-related gene (HERG) potassium channels. Basic Clin. Pharmacol. Toxicol. 2005, 96, 123–130. [CrossRef][PubMed] 15. Ni, Y.; Wang, T.; Zhuo, X.; Song, B.; Zhang, J.; Wei, F.; Bai, H.; Wang, X.; Yang, D.; Gao, L.; et al. Bisoprolol reversed small conductance calcium-activated potassium channel (SK) remodeling in a volume-overload rat model. Mol. Cell. Biochem. 2013, 384, 95–103. [CrossRef][PubMed] 16. Du, Y.; Zhang, J.; Xi, Y.; Wu, G.; Han, K.; Huang, X.; Ma, A.; Wang, T. β1-Adrenergic blocker bisoprolol reverses down-regulated ion channels in sinoatrial node of heart failure rats. J. Physiol. Biochem. 2016, 72, 293–302. [CrossRef][PubMed] 17. Selyanko, A.A.; Hadley, J.K.; Wood, I.C.; Abogadie, F.C.; Delmas, P.; Buckley, N.J.; London, B.; Brown, D.A. Two types of K+ channel subunit, Erg1 and KCNQ2/3, contribute to the M-like current in a mammalian neuronal cell. J. Neurosci. 1999, 19, 7742–7756. [CrossRef] 18. Hsu, H.T.; Tseng, Y.T.; Lo, Y.C.; Wu, S.N. Ability of naringenin, a bioflavonoid, to activate M-type potassium

current in motor neuron-like cells and to increase BKCa-channel activity in HEK293T cells transfected with alpha-hSlo subunit. BMC Neurosci. 2014, 15, 135. [CrossRef] 19. Chen, T.S.; Lai, M.C.; Hung, T.Y.; Lin, K.M.; Huang, C.W.; Wu, S.N. Pioglitazone, a PPAR-γ Activator,

Stimulates BKCa but Suppresses IKM in Hippocampal Neurons. Front. Pharmacol. 2018, 9, 977. [CrossRef] 20. Liu, Y.Y.; Hsiao, H.T.; Wang, J.C.; Liu, Y.C.; Wu, S.N. Parecoxib, a selective blocker of cyclooxygenase-2, directly inhibits neuronal delayed-rectifier K+ current, M-type K+ current and Na+ current. Eur. J. Pharmacol. 2018, 844, 95–101. [CrossRef] 21. Pena, F.; Alavez-Perez, N. Epileptiform activity induced by pharmacologic reduction of M-current in the developing hippocampus in vitro. Epilepsia 2006, 47, 47–54. [CrossRef][PubMed]

22. Migliore, M.; Migliore, R. Know your current Ih: Interaction with a shunting current explains the puzzling effects of its pharmacological or pathological modulations. PLoS ONE 2012, 7, e36867. [CrossRef][PubMed] 23. Kumar, M.; Reed, N.; Liu, R.; Aizenman, E.; Wipf, P.; Tzounopoulos, T. Synthesis and evaluation of potent KCNQ2/3-specific channel activators. Mol. Pharmacol. 2016, 89, 667–677. [CrossRef][PubMed] 24. Du, X.; Gao, H.; Jaffe, D.; Zhang, H.; Gamper, N. M-type K+ channels in peripheral nociceptive pathways. Br. J. Pharmacol. 2018, 175, 2158–2172. [PubMed] 25. Wu, S.N.; Jan, C.R.; Li, H.F.; Chiang, H.T. Characterization of inhibition by risperidone of the inwardly + rectifying K current in pituitary GH3 cells. Neuropsychopharmacology 2000, 23, 676–689. [CrossRef] 26. Furlan, F.; Taccola, G.; Grandolfo, M.; Guasti, L.; Arcangeli, A.; Nistri, A.; Ballerini, L. ERG conductance expression modulates the excitability of ventral horn GABAergic interneurons that control rhythmic oscillations in the developing mouse spinal cord. J. Neurosci. 2007, 27, 919–928. [CrossRef][PubMed] Int. J. Mol. Sci. 2019, 20, 657 19 of 20

27. Chen, B.S.; Lo, Y.C.; Peng, H.; Hsu, T.I.; Wu, S.N. Effects of ranolazine, a novel anti-anginal drug, on ion

currents and membrane potential in pituitary tumor GH3 cells and NG108-15 neuronal cells. J. Pharmacol. Sci. 2009, 110, 295–305. [CrossRef] 28. Carretero, L.; Llavona, P.; López-Hernández, A.; Casado, P.; Cutillas, P.R.; de la Peña, P.; Barros, F.; Dominguez, P. ERK and RSK are necessary for TRH-induced inhibition of r-ERG potassium currents

in rat pituitary GH3 cells. Cell Signal. 2015, 27, 1720–1730. [CrossRef] 29. Huang, M.H.; Shen, A.Y.; Wang, T.S.; Wu, H.M.; Kang, Y.F.; Chen, C.T.; Hsu, T.I.; Chen, B.S.; Wu, S.N. + Inhibitory action of methadone and its metabolites on erg-mediated K current in GH3 pituitary tumor cells. Toxicology 2011, 280, 1–9. [CrossRef] 30. Wu, S.N.; Hsu, M.C.; Liao, Y.K.; Wu, F.T.; Jong, Y.J.; Lo, Y.C. Evidence for inhibitory effects of flupirtine, a centrally acting analgesic, on delayed rectifier K+ currents in motor neuron-like cells. Evid. Based Complement. Alternat. Med. 2012, 2012, 148403. [CrossRef] 31. Colbert, W.E.; Williams, P.D.; Williams, G.D. β-Adrenoceptor profile of ractopamine HCl in isolated smooth and cardiac muscle tissues of rat and guinea-pig. J. Pharm. Pharmacol. 1991, 43, 844–847. [CrossRef][PubMed] 32. Tai, C.T.; Chen, S.A.; Chiang, C.E.; Lee, S.H.; Wen, Z.C.; Chang, M.S.; Wu, S.N. Influence of beta-adrenergic and vagal activity on the effect of exogenous adenosine on supraventricular tachycardia termination. Am. J. Cardiol. 1997, 79, 1628–1631. [CrossRef] 33. Vandenberg, G.; Leatherland, J.; Moccia, R.D. The effects of the beta-agonist ractopamine on growth hormone and intermediary metabolite concentrations in rainbow trout, Oncorhynchus mykiss (Walbaum). Aquac. Res. 2008, 29, 9. [CrossRef] 34. Mills, S.E.; Spurlock, M.E.; Smith, D.J. β-Adrenergic receptor subtypes that mediate ractopamine stimulation of lipolysis. J. Anim. Sci. 2003, 81, 662–668. [CrossRef][PubMed] 35. Stephany, R.W. Hormonal growth promoting agents in food producing animals. Handb. Exp. Pharmacol. 2010, 195, 355–367. 36. Lee, S.H.; Lee, E.H.; Ryu, S.Y.; Rhim, H.; Baek, H.J.; Lim, W.; Ho, W.K. Role of K+ channels in frequency

regulation of spontaneous action potentials in rat pituitary GH3 cells. Neuroendocrinology 2003, 78, 260–269. [CrossRef][PubMed] 37. Hasebe, M.; Oka, Y. High-frequency firing activity of GnRH1 neurons in female Medaka induces the release of GnRH1 peptide from their nerve terminals in the pituitary. Endocrinology 2017, 158, 2603–2617. [CrossRef] [PubMed] 38. Caspi, A.; Benninger, F.; Yaari, Y. KV7/M channels mediate osmotic modulation of intrinsic neuronal excitability. J. Neurosci. 2009, 29, 11098–11111. [CrossRef][PubMed] 39. Chen, P.C.; Ruan, J.S.; Wu, S.N. Evidence of decreased activity in intermediate-conductance calcium-activated potassium channels during retinoic acid-induced differentiation in motor neuron-like NSC-34 cells. Cell. Physiol. Biochem. 2018, 48, 2374–2388. [CrossRef] 40. So, E.C.; Wang, Y.; Yang, L.Q.; So, K.H.; Lo, Y.C.; Wu, S.N. Multiple regulatory actions of 2-guanidine-4-methylquinazoline (GMQ), an agonist of acid-sensing ion channel type 3, on ionic currents

in pituitary GH3 cells and in olfactory sensory (Rolf B1.T) neurons. Biochem. Pharmacol. 2018, 151, 79–88. [CrossRef] 41. Wu, S.N.; Yang, W.H.; Yeh, C.C.; Huang, H.C. The inhibition by di(2-ethylhexyl)-phthalate of erg-mediated + K current in pituitary tumor (GH3) cells. Arch. Toxicol. 2012, 86, 713–723. [CrossRef][PubMed] 42. Wu, S.N.; Chern, J.H.; Shen, S.; Chen, H.H.; Hsu, Y.T.; Lee, C.C.; Chan, M.H.; Lai, M.C.; Shie, F.S. Stimulatory actions of a novel thiourea derivative on large-conductance, calcium-activated potassium channels. J. Cell. Physiol. 2017, 232, 3409–3421. [CrossRef][PubMed] 43. Zhou, J.; Augelli-Szafran, C.E.; Bradley, J.A.; Chen, X.; Koci, B.J.; Volberg, W.A.; Sun, Z.; Cordes, J.S. Novel potent human ether-a-go-go-related gene (hERG) potassium channel enhancers and their in vitro antiarrhythmic activity. Mol. Pharmacol. 2005, 68, 876–884. [PubMed] 44. Yu, H.; Wu, M.; Townsend, S.D.; Zou, B.; Long, S.; Daniels, J.S.; McManus, O.B.; Li, M.; Lindsley, C.W.; Hopkins, C.R. Discovery, synthesis, and structure activity relationship of a series of N-aryl- bicyclo[2.2.1] heptane-2-carboxamides: Characterization of ML213 as a novel KCNQ2 and KCNQ4 potassium channel opener. ACS Chem. Neurosci. 2011, 2, 572–577. [CrossRef][PubMed] Int. J. Mol. Sci. 2019, 20, 657 20 of 20

45. Gatford, K.L.; De Blasio, M.J.; Roberts, C.T.; Nottle, M.B.; Kind, K.L.; van Wettere, W.H.; Smits, R.J.; Owens, J.A. Responses to maternal GH or ractopamine during early-mid pregnancy are similar in primiparous and multiparous pregnant pigs. J. Endocrinol. 2009, 203, 143–154. [CrossRef][PubMed] 46. Momˇcilovi´c,S.; Milovanovi´c,J.R.; Jankovi´c,S.M.; Jovanovi´c,A.; Tasi´c-Otaševi´c,S.; Stanojevi´c,D.; Krsti´c,M.; Šalinger-Martinovi´c,S.; Radojkovi´c,D.D.; Damjanovi´c,M.; et al. Population pharmacokinetic analysis of bisoprolol in patients with acute coronary syndrome. J. Cardiovasc. Pharmacol. 2018. (Epub ahear of print). 47. Janssens, K.; Boussemaere, M.; Wagner, S.; Kopka, K.; Denef, C. β1-Adrenoceptors in rat anterior pituitary may be constitutively active. inverse agonism of CGP 20712A on basal 3’,5’-cyclic adenosine 5’-monophosphate levels. Endocrinology 2008, 149, 2391–2402. [CrossRef] 48. Huang, Y.C.; Wu, B.N.; Lin, Y.T.; Chen, S.J.; Chiu, C.C.; Cheng, C.J.; Chen, I.J. Eugenodilol: A third-generation beta-adrenoceptor blocker, derived from eugenol, with alpha-adrenoceptor blocking and beta2-adrenoceptor agonist-associated vasorelaxant activities. J. Cardiovasc. Pharmacol. 1999, 34, 10–20. [CrossRef] 49. Wu, B.N.; Shen, K.P.; Lin, R.J.; Huang, Y.C.; Chiang, L.C.; Lo, Y.C.; Lin, C.Y.; Chen, I.J. Lipid solubility of vasodilatory vanilloid-type beta-blockers on the functional and binding activities of beta-adrenoceptor subtypes. Gen. Pharmacol. 2000, 34, 321–328. [CrossRef] 50. Almeman, A.A.; Beshir, Y.A.; Aldosary, A.H. Comparison of the effects of metoprolol and bisoprolol on lipid and glucose profiles in cardiovascular patients. Curr. Drug Saf. 2018. (Epub ahead of print). [CrossRef] 51. Fowkes, R.C.; Forrest-Owen, W.; McArdle, C.A. C-type natriuretic peptide (CNP) effects in anterior pituitary cell lines: Evidence for homologous desensitisation of CNP-stimulated cGMP accumulation in alpha T3-1 gonadotroph-derived cells. J. Endocrinol. 2000, 166, 195–203. [CrossRef][PubMed] 52. Zhou, B.R.; Zhou, H.F.; Liu, B.; Jiang, X.F.; Xu, J.Y.; Jiang, M. The effect of Bushen Zhuyun prescription on gonadotropic hormone secretion and key transcription factor expression in rat pituitary cells. Nanosci. Nanotechnol. Lett. 2017, 9, 1596–1603. [CrossRef] 53. Gingerich, S.; Kim, G.L.; Chalmers, J.A.; Koletar, M.M.; Wang, X.; Wang, Y.; Belsham, D.D. Estrogen receptor alpha and G-protein coupled receptor 30 mediate the neuroprotective effects of 17beta-estradiol in novel murine hippocampal cell models. Neuroscience 2010, 170, 54–66. [CrossRef][PubMed] 54. Huang, C.W.; Chow, J.C.; Tsai, J.J.; Wu, S.N. Characterizing the effects of Eugenol on neuronal ionic currents and hyperexcitability. Psychopharmacology 2012, 221, 575–587. [CrossRef][PubMed]

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