Characterization of Inhibitory Capability on Hyperpolarization-Activated Cation Current Caused by Lutein (β,ε-Carotene-3,3′-Diol), a Dietary Xanthophyll Carotenoid

Abstract

Lutein (β,ε-carotene-3,3′-diol), a xanthophyll carotenoid, is found in high concentrations in the macula of the human retina. It has been recognized to exert potential effectiveness in antioxidative and anti-inflammatory properties. However, whether and how its modifications on varying types of plasmalemmal ionic currents occur in electrically excitable cells remain incompletely answered. The current hypothesis is that lutein produces any direct adjustments on ionic currents (e.g., hyperpolarization-activated cation current, I_h [or funny current, I_f]). In the present study, GH₃-cell exposure to lutein resulted in a time-, state- and concentration-dependent reduction in I_h amplitude with an IC₅₀ value of 4.1 μM. There was a hyperpolarizing shift along the voltage axis in the steady-state activation curve of I_h in the presence of this compound, despite being void of changes in the gating charge of the curve. Under continued exposure to lutein (3 μM), further addition of oxaliplatin (10 μM) or ivabradine (3 μM) could be effective at either reversing or further decreasing lutein-induced suppression of hyperpolarization-evoked I_h, respectively. The voltage-dependent anti-clockwise hysteresis of I_h responding to long-lasting inverted isosceles-triangular ramp concentration-dependently became diminished by adding this compound. However, the addition of 10 μM lutein caused a mild but significant suppression in the amplitude of erg-mediated or A-type K⁺ currents. Under current-clamp potential recordings, the sag potential evoked by long-lasting hyperpolarizing current stimulus was reduced under cell exposure to lutein. Altogether, findings from the current observations enabled us to reflect that during cell exposure to lutein used at pharmacologically achievable concentrations, lutein-perturbed inhibition of I_h would be an ionic mechanism underlying its changes in membrane excitability.

1. Introduction

Lutein (xanthophyll, β,ε-carotene-3,3′-diol), derived from a hydride of a (6′R)-β,ε-carotene, is one of the few xanthophyll carotenoids that is believed to exist not only in vegetables and fruits, but also in high concentrations present in the macula of the human retina, where it is thought to act as a yellow filter [1,2,3,4,5]. Evidence from human studies suggests that dietary intake of lutein can lead to the accumulation of lutein in retinal neural tissue, thereby presumably promoting eye and brain health [6]. Moreover, the accumulation of lutein in the retina has been reported to influence the early maturation of the retina [6]. Alternatively, the beneficial bioactive effects of lutein have been widely recognized to be attributable to its antioxidant and anti-inflammatory properties. Indeed, many basic and clinical studies have growingly reported the capability of lutein to exert anti-oxidative and anti-inflammatory properties in the eye, since this compound might act as an antioxidant, protecting cells against the damaging effects of free radicals [2,4,5,6,7,8,9,10,11,12,13,14].

Moreover, lutein cotreatment with antiepileptic drugs was recently reported to be a promising strategy either to improve therapeutic efficacy in patients suffering from epilepsy [15] or to improve cognitive function [16]. This compound has been additionally revealed to ameliorate either ischemia-reperfusion-induced vasculitic neuropathic pain and neuropathy in rats, or photoreceptor degeneration in a mouse model of retinitis pigmentosa [17,18,19]. It was also recently reported to exert anti-neoplastic action [20,21]. Changes in oxidative stress or the activity of NADPH oxidase have been recently demonstrated to affect different types of plasmalemmal ionic currents [22]. However, to our surprise, the ionic mechanism of lutein-induced actions through which it affects the functional activities of excitable cells remains largely unanswered.

The hyperpolarization-activated cation current (I_h or funny current [I_f]) is well-recognized as being a key determinant of repetitive electrical activity in heart cells and in various types of neurons and neuroendocrine or endocrine cells [23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38]. This type of ionic current comprises a mixed inward Na⁺/K⁺ current (i.e., unusual ion selectivity in that it can conduct both Na⁺ and K⁺ ions), which is vulnerable to inhibition by CsCl, ivabradine, or ZD7288 [23,26,39,40,41,42,43]. It is believed that the activation of this current, which proceeds at resting membrane potential, is allowed to produce a net inwardly directed current carried largely by Na⁺ ions, thereby bringing about membrane depolarization to the threshold critically involved in the generation of action potentials [23,25,32,44]. These currents are assumed to be carried by channels of the hyperpolarization-activated cyclic nucleotide-gated (HCN) gene family, which belongs to the superfamily of voltage-gated K⁺ (K_V) channels and cyclic nucleotide-gated (CNG) channels [26,28,34,37,44,45,46]. HCN2, HCN3, or mixed HCN2 + HCN3 channels were reported previously to be distributed in pituitary tumor (GH₃) cells [27,28,39]. The activities of CNG and HCN channels have also been thought to be closely linked to phototransduction intrinsically found in photosensitive retinal ganglion cells [47]. At present, the issue of whether and how lutein can interact with the HCN channel to modify the amplitude, gating properties, and hysteretic magnitude of I_h remains unsolved.

In view of the foregoing considerations, the possible electrophysiological actions of lutein and other related compounds in pituitary GH₃ cells were explored in this study. We tried to study the effects of lutein on the amplitude and gating of I_h residing in pituitary GH₃ somatolactotrophs. Findings from the current observations tempt us to reflect that the I_h present in different cell types could be an unidentified but distinctive target through which the lutein molecule can act to affect the functional activities of the cells involved.

2. Results

2.1. Suppressive Effect of Lutein on Hyperpolarization-Activated Cation Current (I_h) Measured from Pituitary Tumor (GH₃) Cells

In this study, we began to exploit the whole-cell configuration of the patch-clamp technique to evaluate any possible modifications of lutein on ionic currents identified in GH₃ cells. In order to record ionic current flowing through I_h, we kept cells in Ca²⁺-free, Tyrode’s solution containing 0.5 mM CdCl₂ and 1 μM tetrodotoxin (TTX), and the recording pipette that was used was filled up with a K⁺-containing (145 mM). TTX or CdCl₂ was used to block voltage-gated Na⁺ or Ca²⁺ currents, respectively. As the whole-cell mode was firmly achieved, we maintained the tested cell at a holding potential of −40 mV and a 2-sec hyperpolarizing step to −120 mV was thereafter imposed to evoke I_h. Of interest, one minute after cell exposure to lutein, the amplitude of I_h evoked in response to such 2-sec long step hyperpolarization from −40 to −120 mV became progressively lessened (Figure 1A). For example, at the level of −120 mV, the application of the compound at a concentration of 3 μM resulted in a decrease of I_h amplitude from 338 ± 27 to 201 ± 18 pA (n = 7, p < 0.05). After the lutein was removed, the current amplitude was returned to 332 ± 25 pA (n = 7). Concurrently, under GH₃-cell exposure to 3 μM lutein, the value of activation time constant (τ_act) significantly arose from 1.4 ± 0.1 s to 2.8 ± 0.2 s (n = 7, p < 0.05).

The relationship between the concentration of lutein and the amplitude of I_h was further collated, and the results are hence presented in Figure 1B. In this stage of measurements, we held the examined cell in a voltage clamp at −40 mV, and the hyperpolarizing pulse (2-sec duration) to −120 mV was thereafter imposed on it, while current amplitudes during cell exposure to varying lutein concentrations (0.1–100 μM) were measured at the end pulse of long-lasting hyperpolarizing command voltage. It became clear that cell exposure to lutein is able to suppress I_h in a concentration-dependent manner in these cells. By virtue of a sigmoidal least-square fit to the experimental data, the value of effective IC₅₀ (i.e., the concentration required for a half-maximal inhibition) for the inhibitory effect of lutein on I_h was properly yielded at 4.1 μM. Additionally, the existence of lutein increased the τ_act value of I_h evoked by long-lasting membrane hyperpolarization (Figure 1C). The results tempt us to reflect that, under our experimental conditions, lutein can bring about a depressant action of I_h in these cells in a time- and concentration-dependent manner.

2.2. Effect of Lutein on the Quasi-Steady-State Activation Curve of I_h Identified in GH₃ Cells

To characterize the inhibitory effect of lutein on I_h, an effort was made to ascertain if cell exposure to lutein might modify any changes in the steady-state activation curve of I_h intrinsically in GH₃ cells. Figure 2C illustrates the steady-state activation curve of I_h acquired without or with the presence of this compound at a concentration of 3 μM. The Boltzmann distribution (or Fermi-Dirac distribution) detailed in Section 4 was applied to fit the experimental data points appropriately. In the control (i.e., in the absence of lutein), V_1/2 = −82 ± 8 mV and q = 4.35 ± 0.07 e (n = 7), and during the exposure to 3 μM lutein, V_1/2 = −93 ± 9 mV and q = 4.29 ± 0.08 e (n = 7). Consequently, the emerging data led us to project that during GH₃-cell exposure to lutein (3 μM), the steady-state activation curve of I_h elicited under different preceding levels of conditioning step-pulse potential was shifted along the voltage axis toward more hyperpolarized potential (approximately 11 mV), despite no concurrent changes in the apparent gating charge (i.e., q) of the curve.

2.3. Comparisons among the Effect of Lutein, Lutein Plus Oxaliplatin and Lutein Plus Ivabradine on I_h Amplitude

We then investigated if the addition of oxaliplatin or ivabradine, but still in the continued exposure to 3 μM lutein, was able to adjust lutein-perturbed inhibition of I_h identified in GH₃ cells. Oxaliplatin or ivabradine has been recently demonstrated to activate or suppress I_h, respectively [40,48,49]. In this set of current recordings, we placed cells in Ca²⁺-free, Tyrode’s solution containing 1 μM TTX, and the I_h residing in each cell was activated by stepping to −120 mV for a duration of 2 sec from a holding potential of −40 mV. As can be seen in Figure 3A,B, the subsequent addition of 10 μM oxaliplatin or 3 μM ivabradine, but still in continued exposure to 3 μM lutein, was able to attenuate or further decrease I_h amplitude, respectively. It is, therefore, clear that the I_h magnitude susceptible to being inhibited by lutein presented in GH₃ cells would be effectively reversed by further application of oxaliplatin; however, it was further reduced by adding ivabradine.

2.4. Effect of Lutein on Voltage-Dependent Hysteresis (Hys_(V)) of I_h Triggered by Inverted Isosceles-Triangular Ramp Voltage (V_ramp)

Previous observations disclosed the capability of I_h strength evoked by V_ramp to modify the varying patterns of bursting firing in different types of excitable cells, including central neurons [29,35,50,51,52]. Cyclic nucleotide-gated (CNG) channels (e.g., CNGA2 ion channels) were previously demonstrated to exhibit Hys_(V) behavior [53]. We hence continued to evaluate how lutein could have any propensity to adjust I_h’s Hys_(V) responding to long-lasting triangular V_ramp, the protocol of which was designed and then imposed on the tested cells through digital-to-analog conversion. In the current measurements, the tested cell was held at −40 mV, and we then applied a long-lasting inverted V_ramp to it. The V_ramp protocol comprises the downsloping (forward) ramp from −40 to −150 mV followed by the upsloping (backward) limb back to −40 mV with a total duration of 3.2 sec (i.e., a ramp speed of ±69 mV/sec) as indicated in the upper part of Figure 4A. As demonstrated in Figure 4, the voltage-dependent hysteresis (Hys_(V)) of I_h (i.e., the relationship of forward or backward I_h versus membrane potential) was robustly observed upon activation by triangular double V_ramp. That is, the I_h amplitude triggered by the downsloping limb of the inverted triangular V_ramp was overly lower than that by the upsloping end of V_ramp. For example, in the control period (i.e., lutein was not present), the I_h amplitude at −120 mV taken during the downsloping or upsloping end of V_ramp was markedly different (p < 0.05) (i.e., 48 ± 8 pA [downsloping] versus 249 ± pA [upsloping]; n = 7, p < 0.05). Under cell exposure to lutein (3 μM), I_h evoked in the downsloping limb of the long-lasting inverted V_ramp was noticed to decline to a less extent than that measured from the upsloping end of the triangular V_ramp. Moreover, the strength of lutein-induced current inhibition at the downsloping (forward) and upsloping (reverse) limbs of triangular V_ramp differ significantly. As demonstrated by the dashed arrows in Figure 4A, upon the difference (i.e., ∆area) in the shaded area under the curve in the downsloping and upsloping direction, we further quantified the degree of I_h’s Hys_(V) residing in GH₃ cells. The experimental observations disclosed that the amount of Hys_(V) responding to the long-lasting inverted V_ramp became conceivably decreased during cell exposure to lutein. Figure 4B summarized the data demonstrating the effects of lutein and lutein plus ivabradine on the ∆area (i.e., the shaded region in (A)) under such curve. For example, in addition to its depressive action on I_h magnitude, the addition of 3 μM lutein resulted in a reduction in the area responding to inverted triangular V_ramp, as demonstrated by a reduction of ∆area from 17.7 ± 2.4 to 8.8 ± 1.6 mV·nA (n = 7, p < 0.05). After the lutein was removed, the Hys_(V)’s area returned to 17.2 ± 2.3 mV·nA. As cells were continually exposed to 3 μM lutein, subsequent application of 3 μM ivabradine measurably decreased the ∆area of Hys_(V) further.

2.5. Mild Modification by Lutein of A-Type K⁺ Current (I_K(A)) in GH₃ Cells

We also continued to study if the presence of lutein could influence macroscopic K⁺ currents (e.g., I_K(A)) identified in these cells. These experiments were conducted in cells which were bathed in Ca²⁺-free, Tyrode’s solution containing 1 μM TTX, and we filled up the recording pipette by adding K⁺-enriched (145 mM) solution. As depicted in Figure 5A, with cell exposure to lutein at a concentration of 10 μM, the amplitudes of I_K(A) were robustly reduced, which were activated upon different levels of voltage steps with a rapidly activating and slowing inactivating time course of the current [54,55]. The average I–V relationship of peak and sustained I_K(A) in the control period (i.e., lutein was not present) or during cell exposure to 10 μM lutein was collated and is hence illustrated in Figure 5B or Figure 5C, respectively. For example, cell exposure to 10 μM lutein markedly decreased the peak I_K(A) at 0 mV from 1076 ± 122 to 992 ± 112 pA (n = 7, p < 0.05). Therefore, the presence of lutein (10 μM) brought about a mild suppression in the peak and sustained amplitudes of I_K(A) with no marked change in the inactivation time course of the current.

2.6. Effect of Lutein on erg-Mediated K⁺ Current (I_K(erg)) in GH₃ Cells

We also explored whether cell exposure to lutein could modify another type of K⁺ currents (e.g., I_K(erg)). To amplify I_K(erg), we placed cells in a high-K⁺, Ca²⁺-free solution, and the recording pipette was filled with K⁺-containing solution. As shown in Figure 6A, when we held the tested cells at −10 mV, and a 1 sec hyperpolarizing step to −90 mV was imposed to activate the deactivating I_K(erg) with a slowly decaying time course [56,57,58]. One minute after cells were exposed to 10 μM lutein, upon membrane hyperpolarization from −10 to −90 mV, the peak amplitude of I_K(erg) decreased from 891 ± 83 to 771 ± 79 pA (n = 7, p < 0.05). After washout of the compound, I_K(erg) amplitude returned to 885 ± 22 pA (n = 7). The average I–V relationship of peak I_K(erg) with or without cell exposure to 10 μM lutein was constructed and is hence illustrated in Figure 6B. Therefore, similar to the results described above in I_K(A), the data showed that lutein led to a mild inhibitory effect on I_K(erg) activated in response to long-lasting membrane hyperpolarization.

2.7. Effect of Lutein on Sag Potential Measured from GH₃ Cells

We further switched to current-clamp potential recordings in an attempt to examine if lutein could result in any changes on sag potential in these cells. Sag potential evoked in response to hyperpolarizing current stimulus has been demonstrated previously to be linked to the occurrence of I_h in different types of excitable cells [59,60,61]. In this set of experiments, GH₃ cells were placed in normal Tyrode’s solution containing 1.8 mM CaCl₂, and the pipette was filled with K⁺-enrich solution. Current-clamp configuration with a holding current of 0 pA was then established. As shown in Figure 7, under our experimental conditions, when the whole-cell potential recordings were achieved, a long-step 2-sec hyperpolarizing current injection with the amplitude of around 25 pA was strikingly noticed to induce the occurrence of sag potential (i.e., an abrupt drop-down to a lower level in the membrane potential upon hyperpolarizing current injection) [62]. Cell exposure to ivabradine (3 μM) was effective at suppressing the amplitude of sag potential. Furthermore, the application of 1 or 3 μM lutein resulted in a conceivable depression of sag potential evoked in response to hyperpolarizing current stimulus. For example, lutein at a concentration of 3 μM measurably decreased the amplitude of sag potential from 43.5 ± 7.0 to 20.5 ± 34.9 mV (n = 7, p < 0.05). It is conceivable, therefore, that the sag potential presented in GH₃ cells is associated with the magnitude of I_h and that the depression of such potential produced by the presence of lutein could be largely ascribed from its inhibitory effectiveness in the I_h amplitude of these cells.

3. Discussion

The principal findings in this work are that GH₃-cell exposure to lutein is capable of suppressing I_h in a concentration-, state-, voltage-, and Hys_(V)-dependent manner. A hyperpolarizing shift of the steady-state activation curve of I_h was observed in the presence of this compound. The strength of V_ramp-evoked Hys_(V) was also decreased by adding it. Under current-clamp configuration, the amplitude of sag potential observed in GH₃ cells was also decreased during exposure to lutein. HCN2, HCN3, or mixed HCN2+HCN3 channels that can be encoded for generation of macroscopic I_h were reported to be distributed in pituitary GH₃ cells [27,28,39]. Mild inhibition of I_K(erg) or I_K(A) by adding lutein was observed in this study. Under the current-clamp configuration, the exposure to lutein can result in a decrease in the magnitude of sag potential. Together, findings from the present study can thus be interpreted to mean that, besides its antioxidative or anti-inflammatory properties, the existence of lutein can inhibit the amplitude as well as alter gating and Hys_(V) behavior of I_h, and that lutein’s actions presented herein would engage in the modifications on spontaneous actions potentials present in electrically excitable cells (e.g., GH₃ cells), presuming that similar in vivo observations occur.

In this study, a left shift in the steady-state activation curve of I_h with no concurrent modifications in the number of apparent gating charge in the curve was demonstrated with the existence of lutein. The τ_act value estimated from the slowly activating I_h during cell exposure to lutein also turned out to be larger because of a concurrent slowing in the activation time course of the current. Cell exposure to lutein was thus capable of altering hyperpolarization-activated I_h in a time-, concentration- and voltage-dependent manner. Whatever the ionic mechanism of the active lutein, these results can be interpreted to mean that the responsiveness in different cell types to this compound would heavily rely either on the lutein concentration achieved, pre-existing levels of resting membrane potential, different patterns of action potential firing, or a combination of the three.

The Hys_(V) properties of I_h activated in response to triangular V_ramp have been viewed to play a role in perturbing the electrical behaviors of various excitable cells [29,38,50,51,53,63]. Voltage-sensing domain relaxation has also been noticed to have a unique impact on such Hys_(V) behavior observed in the proteins (i.e., HCNx channels) [29,50,64,65]. In other words, the observed “inertia” in the responsiveness of HCNx channels can be driven by changes in their electrical sensitivity, which was allowed to resemble that of ferromagnetic materials displaying Hys_(V) behaviors [52,66]. Of considerable interest, in accordance with previous observations [29,49,63], the I_h intrinsically residing in GH₃ cells was noticed to undergo a non-equilibrium property of instantaneous I_h—that is, an anti-clockwise Hys_(V) loop responding to the isosceles-triangular V_ramp. Such change has been viewed to be dynamically linked to a mode shift in situations where the voltage sensitivity of gating charge movements (i.e., voltage-sensing domain relaxation) relies on the previous state (or conformation) of the channel [50,52,63,64]. Notably, GH₃-cell exposure to lutein presented herein resulted in a significant reduction in Hys_(V) strength (i.e., shaded region in Figure 4) of I_h evoked by long-lasting triangular V_ramp. However, whether the lutein molecule can interact with the voltage-sensing domains of HCNx channels remains to be further delineated.

In previous studies, different HCN isoforms have been demonstrated to combine to constitute macroscopic I_h existing in different types of excitable cells [26,28,67]. Because HCN2, HCN3, or mixed HCN2+HCN3 channels were reported to be functionally expressed in GH₃ cells [28,39], it appears unlikely that lutein-induced block of I_h inherently in native cells is isoform-specific. Of additional note, it has been reported that the blocker of HCN channels could be linked to changes in the phosphene perception of the retina [68], which might potentially account for lutein-mediated amelioration in the retina (e.g., macular degeneration) [4,7,8,10,37].

Earlier investigations have shown that the cyclic nucleotide-gated (CNG) channels, the behavior of which was noticed to show Hys_(V) behavior in their gating mechanism, are regarded to mediate receptor potentials involved in olfaction and vision [53]. The HCN channels, another family of ionic channels gated by cyclic nucleotides, have been previously demonstrated to be linked to phototransduction in photosensitive retinal ganglion cells [47]. Its activity was found to alter the electroretinographic ON and OFF responses or to delay photoreceptor degeneration [19,68,69,70]. To what extent do lutein-mediated changes in magnitude, gating kinetics and Hys_(V) behavior of I_h presented herein engage in lutein-mediated action on age-related diseases (e.g., macular degeneration or retinitis pigmentosa) [8,19,71,72,73,74] still needs to be further delineated.

In this study, the lutein concentration required for the half-maximal inhibition (i.e., IC₅₀) of I_h detected in GH₃ cells was calculated to be 4.1 μM. From the previous pharmacokinetic studies, it has been previously reported that plasma lutein concentration could reach around 2.5 μM after daily doses of lutein (20.5 mg) for 42 days, and that it could increase 3.5- and 10-fold on average, respectively, after the long-term intake of 4.1 and 20.5 mg lutein [75]. In comparison, it is, therefore, reasonable to assume that the observed effects by lutein on the amplitude, gating and Hys_(V) of I_h (or HCNx-encoded currents) may achieve the concentration of therapeutic or pharmacological requirements [75,76].

In this study, we additionally investigated how the protein of HCN channel could be docked with the lutein molecule through PyRx software. The protein structure of HCN channel was acquired from PDB (PDB ID: 5V4S) [77]. The predicted binding sites of the lutein with which the amino-acid residues can interact are presented in Figure 8. It should be noted that the lutein molecule may form hydrophobic contacts with residue Leu240(A), Leu240(D), Ala241(A), Ala241(C), Ala243(B), Ala243(D), Ala244(A), Ala244(D), Arg246(D), Lys247(D), Ala250(B), Ala250(D), and Gln251(B), while it has a hydrogen bond with residue Ser254(B) with a distance of approximately 3.07 Å. These results prompted us to reflect that the lutein molecule can bind to the transmembrane segment (position: 242–269) of HCN channels (PDB: 5V4S), the site of which is different to a certain extent from those reported recently [43]. It is thus likely that the interaction of the lutein molecules with HCN channels could be located at the cytosolic side of the membrane, although the detailed ionic mechanisms of lutein’s actions are currently unknown.

4. Materials and Methods

4.1. Chemicals, Drugs and Solutions Used in the Present Work

Lutein (xanthophyll, Yè huáng sù, β,ε-carotene-3,3′-diol, 3,3′-di-hydroxy-β,ε-carotene, (1R)-4-[(1E,3E,5E,7E,9E,11E,13E,15E,17E)-18-[(1R,4R)-4-hydroxy-2,6,6-trimethylcyclohex-2-en-1-yl]-3,7,12,16-tetramethyloctadeca-1,3,5,7,9,11,13,15,17-nonaenyl]-3,5,5-trimethylcyclohex-3-en-1-ol, CID 5281243, C40H56O2, https://pubchem.ncbi.nlm.nih.gov/compound/5281243) (accessed on 1 June 2022) was acquired from MedChemExpress (Genechain, Kaohsiung, Taiwan), while ivabradine, oxaliplatin and tetrodotoxin (TTX) were from Sigma-Aldrich (St. Louis, MO). Lutein was dissolved in dimethyl sulfoxide at a concentration of 10 mM and made immediately before experiments. To protect lutein from being degraded by light [7], stock solution containing this compound was wrapped in aluminum foil, and it was kept under −20 °C for long-term storage. For cell preparations, we acquired fetal bovine and calf serum, L-glutamine, trypsin/EDTA, and cell culture media from HycloneTM (Thermo Fisher; Level Biotech, Tainan, Taiwan). Unless stated otherwise, other chemicals, reagents, or solvents were of analytical reagent grade and supplied by Sigma-Aldrich.

The HEPES-buffered normal Tyrode’s solution contained (in mM): NaCl 136.5, KCl 5.4, MgCl₂ 0.53, CaCl₂ 1.8, glucose 5.5, HEPES 5.5, and the pH was adjusted to 7.4 by adding NaOH. For measurements of I_h or I_K(A) across the cell membrane, GH₃ cells were placed in Ca²⁺-free, Tyrode’s solution in order to avoid the contamination of voltage-gated Ca²⁺ currents and Ca²⁺-activated K⁺ currents. To record I_K(erg), we bathed cells in high-K⁺, Ca²⁺-free solution containing: KCl 145, MgCl₂ 0.53 and HEPES-KOH 5 (in mM and pH 7.4). In whole-cell current or potential recordings, we filled up the recording pipette with an internal solution, which contained: K-aspartate 140, KH₂PO₄ 1, Na₂ATP 3, Na₂GTP 0.1, EGTA 0.1, and HEPES 4 (in mM), and the pH was adjusted to 7.4 with KOH. The chemicals or reagents used to make these solutions were acquired from Sigma-Aldrich. The twice-distilled water was deionized through a Millipore-Q system and used in all experiments.

4.2. Cell Preparations

Pituitary GH₃ somatolactotrophs, originally supplied by the Bioresources Collection and Research Center ([BCRC-60015, http://catalog.bcrc.firdi.org.tw/BcrcContent?bid=60015] (accessed on 1 June 2022), Hsinchu, Taiwan), were maintained in Ham’s F-12 medium, with which 15% heat-inactivated horse serum (v/v), 2.5% fetal calf serum (v/v), and 2 mM L-glutamine were supplemented. They were grown in monolayer culture in 50-mL plastic culture flasks in a humidified environment of CO₂/air (1:19). Subcultures were made by trypsinization (0.025% trypsin solution [HyClone^TM] containing 0.01% sodium N,N-diethyldithiocarbamate and EDTA). We carried out electrophysiological measurements 5 or 6 days after GH₃ cells underwent subculture (60–70% confluence).

4.3. Electrophysiological Measurements

Shortly before the recordings, we carefully dispersed the GH₃ with 1% trypsin-EDTA solution, and thereafter placed a few aliquots of cell suspension containing clumps of cells into a home-made chamber tightly mounted on the working stage of a DM-II inverted microscope (Leica; Major Instruments, Tainan, Taiwan). Cells were immersed at room temperature (20–25 °C) in HEPES-buffered normal Tyrode’s solution, ionic composition of which was elaborated above. The electrode that we used to record were prepared from Kimax^®-51 capillaries with 1.5–1.8 mm outer diameter (#34500; Kimble; Dogger, New Taipei City, Taiwan) by using a vertical puller (PP-83; Narishige, Major Instruments). Being filled with different internal solutions stated above, the recording electrodes had tip resistances of 3–5 MΩ. We measured ionic currents or membrane potential in the whole-cell current or potential recording of a modified patch-clamp technique, respectively, by using either a MultiClamp 700B (Molecular Devices, Sunnyvale, CA) or an RK-400 amplifier (Bio-Logic, Claix, France), as described elsewhere [24,49,55]. GΩ-seals were established in an all-or-nothing fashion and hence resulted in a dramatic improvement in the signal-to-noise ratio. The liquid junction potential, which occurred when the ionic compositions in the pipette solution and those of the external solution were different, was zeroed shortly before GΩ-seal formation was made, and the whole-cell data were then corrected. During measurements, we exchanged the solution through a homemade gravity-driven type of bath perfusion. In order to measure the I_h, we voltage-clamped the examined cell at a holding potential of −40 mV before a long-lasting hyperpolarizing step was delivered.

The signals, comprising both potential and current traces, were monitored at a given interval and digitally stored online at 10 kHz in an ASUS ExpertBook laptop computer (P2451F; Yuan-Dai, Tainan, Taiwan). For efficient analog-to-digital (A/D) and digital-to-analog (D/A) conversion to proceed during the measurements, a low-noise Digidata^® 1440A equipped with an ASUS computer was controlled by pClamp^TM 10.6 software run under Microsoft Windows 7 (Redmond, WA, USA). We low-pass filtered current signals at 2 kHz with a FL-4 four-pole Bessel filter (Dagan, Minneapolis, MN, USA). A variety of pClamp-generated voltage-clamp protocols with various rectangular or ramp waveforms were designed, and they were then applied to the tested cells through D/A conversion in attempts to assess the current–voltage (I–V) relation or steady-state activation curve of the currents.

4.4. Data Analyses

To assess concentration-dependent inhibition of lutein on I_h amplitude, we put GH₃ cells in Ca²⁺-free Tyrode’s solution. During the measurements, we voltage-clamped each cell at a holding potential of −40 mV and a long-lasting hyperpolarizing pulse to −120 mV for a duration of 2 sec was delivered to evoke I_h. The I_h amplitudes were thereafter measured at the end pulse of 2-sec hyperpolarizing command voltage during cell exposure to different lutein concentrations (0.1–100 μM). The concentration-dependent inhibition by lutein of I_h in GH₃ cells was determined by fitting a modified Hill function. That is,

$$\mathrm{Percentage}\mathrm{decrease}\left(\%\right)=\raisebox{1ex}{$\left(E_{max}\times {\left[lutein\right]}^{n_H}\right)$}\!\left/ \!\raisebox{-1ex}{$\left(I{C}_{50}^{n_H}+{\left[lutein\right]}^{n_H}\right)$}\right.$$

where [lutein] = the lutein concentration applied; n_H = the Hill coefficient; IC₅₀ = the concentration required for a 50% inhibition of I_h amplitude activated by long-lasting hyperpolarizing step; E_max = lutein-induced maximal inhibition of I_h.

To assess lutein-induced modifications on the quasi-steady-state activation of I_h, the relationship between the normalized amplitude of I_h and the conditioning potential acquired with or without cell exposure to 3 μM lutein was constructed, and the data were thereafter fitted by chi-squared goodness of fit with a Boltzmann function of the following form:

$$I=\raisebox{1ex}{$I_{max}$}\!\left/ \!\raisebox{-1ex}{$\left\{1+exp\left[-\left(V-V_{\frac{1}{2}}\right)RT/qF\right]\right\}$}\right.$$

where I_max = the maximal I_h magnitude acquired with or without cell exposure to the lutein (3 μM) at the conditioning potential of −30 mV; V_1/2 = the voltage at which half-maximal activation (i.e., midpoint potential) occurs; q = the apparent gating charge; F = the Faraday constant, R the idea gas constant; T = the absolute temperature.

4.5. Curve-Fitting Approximations and Statistical Analyses

Curve-fitting (linear or non-linear [e.g., Hill or Boltzmann equation] to experimental data sets was dealt with the goodness of fit by using different analytical procedures, which include the Microsoft “Solver” built into Excel^® 2019 (Microsoft) and OriginPro^® 2021 program (Microcal; Scientific Formosa, Kaohsiung, Taiwan). The electrophysiological data (whole-cell current or potential data) are presented as the mean ± standard error of the mean (SEM), with the size of experimental observations (n) indicative of cell numbers from which the data were obtained. The data distribution was found to satisfy the tests for normality. We performed Student’s t-tests (for paired or unpaired samples) between the two different groups, while for more than two different groups, analysis of variance (for one-way ANOVA or two-way ANOVA) with or without repeated measured followed by a post-hoc Fisher’s least-significance difference test was utilized to determine the significance. A p-value of less than 0.05 was considered to indicate statistical difference (marked with * or ** in the figures).

5. Conclusions

These results reveal that lutein profoundly leads to a reduction in hyperpolarization-evoked I_h (or HCNx-encoded currents) in a concentration-, time-, voltage-, and in a Hys_(V)-dependent manner. Our findings on GH₃ cells thus shed light on the evidence revealing the effectiveness of lutein in modifying specific ionic currents (e.g., I_h), and these data may be linked to its additional effects, which are potentially useful pharmacological applications in different excitable cells occurring in vivo.