Recent Advances in Ionic Mechanisms in Pituitary Cells: Implications for Electrophysiological and Electropharmacological Research

1. Introduction

The pituitary gland, often referred to as the “master gland”, is essential for regulating numerous physiological processes in the body by releasing release hormones. It consists of two main sections, the anterior and posterior pituitary. The anterior pituitary contains various cell types, including somatotrophs, thyrotrophs, corticotrophs, gonadotrophs, melanotrophs, and lactotrophs. Among these, lactotrophs are a specific cell population responsible for secreting prolactin.

By utilizing specialized separation techniques [52], it is possible to immortalize pituitary cells derived from pituitary tumors, enabling in-depth academic research into the functional properties of these cells. Additionally, unlike conventional intracellular recordings that involve impaling or piercing the cell, the patch-clamp technique, which uses a relatively large electrode tip with a pipette resistance of 2-4 MΩ, can be used on small cells with minimal damage, making it suitable for cellular electrophysiological experiments [46,121]. In order words, the electrode used in patch-clamp recordings applies negative pressure to generate suction, which holds the small cell, such as pituitary cell, in place or keeps in a relatively suspended position without displacing or distorting it. This method eliminates the need to impale or puncture the cell, reducing cell damage and enhancing electrophysiological recordings. Figure 1 illustrates a typical population of pituitary tumor cells, specifically GH₃ cells.

In this paper, we review the main ion currents in pituitary cells that are influenced by changes in cell membrane voltage, exploring their physiological and pharmacological implications. Figure 2 depicts the major ionic currents commonly observed in pituitary cells. We also provide examples of various drugs or compounds that affect the amplitude and gating kinetics of these ion currents.

Physiological importance and pharmacological impact on voltage-gated ionic currents in pituitary cells

In this review paper, we will provide a detailed description of the potential voltage-gated currents that may occur inn pituitary cells and discuss various drugs or compounds that are currently known to influence these currents (Table 1).

A. Voltage-Gated Na⁺ Current (I_Na)

The Na_V channels exist in nine isoforms (Na_V1.1-1.9, also known as SCN1A-SCN5A and SCN8A-SCN11A) and are found in various mammalian excitable tissues, including the central and peripheral nervous systems, as well as the neuroendocrine or endocrine systems [17,43,110,122]. When activated, Na_V channel activity generates a macroscopic I_Na, which is characterized by its rapid activation and inactivation, both occurring within milliseconds. This current temporarily depolarizes the membrane, providing positive feedback that triggers the upstroke of the action potential (AP). As a result, changes in the magnitude of I_Na can influence the amplitude, frequency, and patterns of APs in various electrically active cells [17,122]. Previous studies have shown that Na_V channels are present in all secretory pituitary cells, including pituitary GH₃ cells [122]. Additionally, late (or persistent) I_Na has been progressively identified, and its presence exerts a significant influence on electrical behavior and firing patterns of pituitary cells [21,30,56,122,143].

• 1. GV-58 ((2R)-2-[(6-{[(5-methylthiophen-2-yl)methyl]amino}-9-propyl-9H-purin-2-yl)amino]butan-1-ol)

GV-58 was developed as a modification of (R)-roscovitine. It has been viewed as an opener of N- and P/Q-type Ca²⁺ channels [12,81,92,126]. This compound was presumably thought to slow the closing of the voltage-gated Ca²⁺ (Ca_V) channel, resulting in a large increase in total Ca²⁺ entry during motor nerve AP activity [126]. Its presence was reported to enhance spontaneous and evoked activity from the cultures of murine ventral horn of the spinal cord on microelectrode arrays [12]. Earlier studies have demonstrated that this compound is effective for the management of neuromuscular weakness, such as Lambert-Eaton myasthenic syndrome [27, 81, 92; 101, 117, 125].

However, recent studies have demonstrated that, as pituitary GH₃ cells were continually exposed to GV-58, the peak and late components of I_Na activated by abrupt step depolarization were increased in a concentration-, time-, and state-dependent manner [27]. The I_Na activated by brief depolarizing pulse was sensitive to either block by tetrodotoxin or stimulation by GV-58, but it failed to be affected by ω-conotoxin MVIID. ω-Conotoxin MVIID, a small, disulfide-rich peptide purified from the venoms of predatory cone snails, was reported to be an inhibitor of N- and P/Q-type Ca²⁺ currents in adrenal chromaffin cells [40]. The recovery of I_Na inactivation induced with varying interpulse intervals was overly enhanced in the presence of GV-58. The decline of peak I_Na during rapid repetitive stimuli was slowed during cell exposure to GV-58. This compound stimulated peak I_Na in a tonic and use-dependent manner. GH₃-cell exposure to GV-58 was also found to enhance the magnitude of instantaneous resurgent and window I_Na. Under current-clamp conditions, GV-58 effectively increased the frequency and spontaneous APs. The NSC-34 motor neuron-like cells could be enhanced by adding GV-58. The mRNA transcripts for the α-subunit of Na_V1.1, Na_V1.2, and Na_V1.6 were reported to be expressed in pituitary GH₃ cells [122]. Findings from this study can be interpreted to reflect that GV-58 can interact with Na_V channels to stimulate the magnitude and to alter the gating of I_Na [27]. These results would engage in the modification of spontaneous APs in electrically excitable cells like GH₃ and NSC-34 cells, presuming that similar in vitro or in vivo findings occur.

Concentration-dependent stimulation of both peak and late I_Na was observed with GV-58, and it exhibited effective half-maximal concentration (EC₅₀) values of 8.9 and 2.6 μM for peak and late I_Na, respectively [27]. Continuous exposure to GV-58 resulted in an increase in the amplitude of peak I_Na in response to abrupt step depolarization, accompanied by a significant increase in the value of slow component of I_Na inactivation time constant. It is therefore important to emphasize caution when ascribing the expanding use of GV-58 to its selective agonistic effects on N- and P/Q-type Ca_V channels [92,101,117].

2.

Esaxerenone (ESAX, Minnebro^®, CS-3150, XL-550, (4S)-4-(5,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)-2-fluoro-N-(1-methyl-1H-pyrazol-4-yl)benzamide)

ESAX, known to be a newly oral, non-steroidal selective blocker on the activity of mineralocorticoid receptor, has been growingly used for the management of varying pathologic disorders, such as primary aldosteronism, refractory hypertension, chronic kidney disease, diabetic nephropathy, and heart failure [5,64,77,98,148]. Alternatively, the activity of mineralocorticoid receptor has been previously demonstrated in pituitary cells including GH₃ cells or in various brain regions [32,41,82,105].

However, earlier studies have shown the ability of ESAX to suppress the peak and late component of I_Na elicited during short depolarizing pulse in pituitary GH₃ cells [21]. The subsequent addition of ESAX reversed tefluthrin-mediated increase in the strength of voltage-dependent hysteresis of persistent I_Na activated by the isosceles-triangular ramp pulse. Tefluthrin was known to be an activator of I_Na [83]. In pituitary MMQ cells, the presence of ESAX was effective at decreasing the amplitude and gating of I_Na.

The IC₅₀ value needed for ESAX-mediated inhibition of peak or late I_Na observed in GH₃ cells was yielded to be 13.2 or 3.2 μM, respectively, the value of which was distinguishable between its suppressive effects on these two components of the current [21]. The presence of neither dexamethasone nor aldosterone affected the magnitude or gating of I_Na. In continued exposure to aldosterone, further addition of ESAX was still able to suppress peak I_Na. It therefore seems unlikely that ESAX-mediated inhibition of I_Na amplitude together with changes in the gating kinetics of the current was predominantly associated with its blockade of mineralocorticoid receptor. Moreover, as shown in Figure 3, the presence of ESAX (10 μM) can effectively diminish the strength in voltage-dependent hysteresis of persistent I_Na (I_Na(P)) activated by prolonged isosceles-triangular ramp voltage observed in GH₃ cells (21].

B. erg-Mediated K⁺ Current (I_K(erg))

The erg (ether-à-go-go related gene)-mediated K⁺ current (I_K(erg)), which is encoded by three different subfamilies of the KCNH gene, is enabled to generate the pore-forming α-subunit of erg-mediated K⁺ (i.e., K_erg or K_V11) channels [9,51]. These K⁺ currents are widely believed to represent the cloned equivalent of the rapidly activating delayed-rectifying K⁺ currents found in cardiac myocytes, and the KCNH2 gene encodes the α-subunit responsible for pore formation in K_V11.1 channels, commonly referred to as the human erg K⁺ (HERG) channels [20,129]. The intrinsic presence of I_K(erg) extends beyond excitable cells to various types of epithelial or neoplastic cells, as reported previously [17,129]. Earlier work has also shown the effectiveness of I_K(erg) magnitude in regulating the apoptosis and proliferation of various types of neoplastic or stem cells [152].

1

Risperidone (Risperdal^®, 3-[2-[4-(6-fluoro-1,2-benzisoxazol-3-yl)piperidin-1-yl]ethyl]-2-methyl-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidin-4-one)

Risperidone, a benzisoxazole compound that has been approved for use in the United States in 1994, is known to be effective at terminating acute psychotic episodes and preventing recurrence of psychotic episodes in schizophrenics [11,13]. However, the significant and consistent neuroendocrine effect of neuroleptic drugs including risperidone is to stimulate prolactin secretion and cause galactorrhea, although these untoward effects vary greatly in potency and chemical structure [13,78,123]. The important site of action has been thought to be due to the blockade of dopamine D2 and 5HT receptors [11,93]. However, other evidence suggests that these neuroleptics, including risperidone, may cause a significant prolongation of electrocardiographic QTc interval [18,118].

In a previous study made in pituitary GH₃ cells, the exposure to risperidone effectively suppressed the magnitude of I_K(erg), together with a slowing in the rate of activation [139]. The results also showed that there was the difference in reciprocal time constants of I_K(erg) decays observed at different voltages, suggesting that risperidone may increase the rate of deactivation [139]. It is therefore possible that the different level of membrane potential can exert an interaction with erg-mediated K⁺ (K_erg) channels to modify the magnitude and gating of whole-cell I_K(erg) in GH₃ cells. In other words, the sensitivity to risperidone in pituitary lactotrophs would be dependent on the preexisting level of resting membrane potential, the firing rate of AP, or the concentration of risperidone used, assuming that the risperidone action in pituitary lactotrophs is similar to that on GH₃ cells [139].

It is worth noting that neither dopamine nor metoclopramide affected the magnitude of I_K(erg) in GH₃ cells, although haloperidol or thioridazine mimicked the risperidone-mediated inhibition of I_K(erg). Metoclopramide was reported to antagonize at dopamine receptor. In GH₃ cells preincubated with dopamine, the inhibitory effect of risperidone on I_K(erg) remained unaffected. Several tyrosine kinase inhibitors were also found to suppress the I_K(erg) magnitude [53]. Therefore, the effect of risperidone on I_K(erg) appear to be direct and independent of its binding to dopamine receptors. The carefulness may need to be made in ascribing the risperidone-mediated prolactin release or QT prolongation to those caused by the blockade of dopamine receptors residing in vivo or in vitro [11,16,75]. These findings imply that the risperidone-mediated stimulation of prolactin release could be partly, if not entirely, ascribed to the direct blockade of I_K(erg) functionally expressed in pituitary lactotrophs [75,123,139].

2

Di(2-ethylhexyl)-phthalate (DEHP)

Phthalates are a group of chemicals that are mainly used as plasticizers to allow stiff plastics, such as polyvinyl chloride, to become more flexible. Because phthalate plasticizers are not chemically bound to polyvinyl chloride, they may leach, migrate, or evaporate into air and atmosphere, foodstuffs, and other materials [3,70]. Due to their suitable properties and low cost, the general population will become significantly exposed to these compounds [63]. One of the phthalate plasticizers used in a wide variety of medical devices is di(2-ethylhexyl)-phthalate (DEHP), which is recognized to be an endocrine-disrupting chemical [42,94,128,134,154].

Previous work has shown that high doses of DEHP could change cell size or function in anterior pituitary gland [94,154]. DEHP was demonstrated to suppress tamoxifen-induced apoptosis possibly linked to its estrogenic effects as well as to influence signaling pathways in pituitary GH₃ cells [38,66]. This compound was also noted to cause an age-dependent influence on the pituitary-adrenocortical axis in vivo [154]. A previous report unveiled that the concentration of phthalate esters, including DEHP, in semen or serum samples were positively associated with circulating prolactin levels in adult men [79]. DEHP was found to impair the electrical and mechanical behavior of cardiac cell network [44].

The study conducted by Wu et al. [142] revealed that DEHP has been observed to reduce the amplitude of I_K(erg) in pituitary GH₃ cells in a concentration-dependent manner, with an IC₅₀ value of 16.3 μM. This IC₅₀ value is notably lower than the typical concentration of DEHP found in human blood or blood components, which has been reported to range from 10 to 650 μg/ml (equivalent to 27 μM to 1.6 mM) [59]. Exposure of GH₃ cells to DHEP was found to alter the activation kinetics of I_K(erg) without affecting in the deactivation kinetics of the current. Additionally, the presence of DEHP led to an increase in the firing of spontaneous APs in these pituitary cells.

We conducted a more in-depth exploration of the interaction between the HERG protein and DEHP using PyRx software. Figure 4 illustrates the predicting docking sites of the DEHP molecule. Notably, during the docking process with the HERG channel, DEHP was noted to establish a hydrogen bond with residue Arg537 with a distance of 3.03 Å. Furthermore, DEHP exhibited hydrophobic interactions with several residues, including Lys407, Asp411, Asn470, Thr474, His492, Tyr493, Trp497, Arg541, Lys538, Asp540, and Arg541. These findings suggest a strong binding affinity between DEHP and the amino acid residues of the HERG channel, estimated at -5.9 kcal/mol. This interaction predominantly occurs in the vicinity of the transmembrane region, specifically around positions 496-516 or 521-541 of the channel. The predicted interaction raises concerns about the potential impact on DEHP-mediated alterations in the magnitude and gating kinetics of I_K(erg).

Taken together, these results suggest that the inhibition of K_erg channels by DHEP or other chemically related compounds may play a role in the changes observed in the functional activities of pituitary cells, including processes like hormonal release, assuming that similar effects can be replicated in a live, in vivo context [142].

C. M-Type K⁺ Current (I_K(M))

The KCNQ2, KCNQ3, and KCNQ5 genes are responsible for encoding the core subunits of K_V7.2, K_V7.3, and K_V7.5 channels, respectively [1,15]. These K⁺ channels, when activated, give rise to the I_K(M), which is found in various electrically excitable cells, including pituitary cells [15,113,144]. This current is characterized by its low threshold voltage activation and displays a slow activation and deactivation profile [15,113]. The regulation of I_K(M) has garnered considerable interest as an adjunctive therapeutic strategy for addressing neurological disorders marked by excessive neuronal activity. These disorders encompass conditions such as cognitive dysfunction, neuropathic pain, and epilepsy [90]. Furthermore, it is believed that the magnitude of I_K(M) play a role in regulating the availability of Na_V channels during extended periods high-frequency firing [150,153].

• 1. Solifenacin (SOL, Vesicare^®, (R)-1-phenyl-3-(1-piperidin-4-ylpropyl)oxy-1,1-diphenyl-4-ylbutan-1-amine)

SOL, a member of isoquinoline, has been viewed as an oral anticholinergic (i.e., a competitive muscarinic [M₁ and M₃] receptor antagonist) and antispasmodic agent used to treat the symptoms of overactive bladder, neurogenic detrusor overactivity, or urinary incontinence [69,89,108]. It has been reported to be a muscarinic (M₂ and M₃) receptor antagonist that has anticholinergic effects such relaxation of the detrusor muscle in urinary bladder [2].

Earlier clinical investigations have revealed the efficacy and safety of the antimuscarinic, SOL, for treating patients with overactive bladder or neurogenic detrusor overactivity [2,69]. However, recent evidence has been reported to demonstrate that the treatment with SOL could be linked to an increased risk of the impairment in cognitive functions [48,135]. It is therefore pertinent to reappraise the mechanism of SOL actions on electrical behaviors in varying excitable cells, given that its growing clinical use occurs.

Many types of anterior pituitary cells have been shown to secrete acetylcholine [72]. Earlier studies have also revealed that pituitary GH₃ cells could exhibit the activity of muscarinic receptors and that muscarinic agonists were able to inhibit hormonal secretion through a reduction in intracellular cyclic AMP [114]. In these pituitary cells, the binding of acetylcholine to M₂-muscarinic receptor might induce a weak stimulation on the hydrolysis of phosphatidylinositol 4,5-bisphosphate [4].

A previous report has shown that in pituitary GH₃ cells, during exposure to SOL, the I_K(M) amplitude elicited upon membrane depolarization was concentration-dependently increased with an EC₅₀ value of 0.34 μM [28]. The activation time course of I_K(M) concurrently became shorted, and the value of dissociation constant (K_D) obtained on the basis of minimal reaction scheme was estimated to 0.55 μM. The value of EC₅₀ required for SOL-mediated effect on I_K(M) was similar, reflecting that SOL has the propensity to bind to the open or activated state of the channel. There was a leftward shift of the quasi-steady-state activation curve of I_K(M) in the presence of SOL. The strength of voltage-dependent hysteresis of I_K(M) activated by isosceles-triangular ramp pulse became increased during cell exposure to SOL. Furthermore, the K_M-channel activity was elevated by the addition of SOL, without affecting single channel conductance of the channel. However, the mean open time of KM channel after exposure to SOL was increased. Under current-clamp conditions, the firing frequency of spontaneous APs present in GH₃ cells was found to be effectively decreased in the presence of SOL. Collectively, findings from these results provide an unanticipated and yet non-canonical ionic mechanisms through which the SOL molecule can interact with K_M channel to enhance whole-cell I_K(M) and, consequently, to diminish the firing rate of spontaneous APs [28]. It turns out that whether the action of SOL or other structurally similar compounds like darifenacin) on overactive bladder or neurogenic detrusor overactivity [2,89,103] are related to its enhanced actions on K_M-channel activity, warrants further investigations, despite its high-affinity binding to muscarinic receptors [2].

2.

Kynurenic acid (KYNA, 4-hydroxyquinoline-2-carboxylic acid)

KYNA is a naturally occurring product of the normal metabolism of amino acid L-tryptophan that has been reported to inhibit N-methyl-D-aspartate receptor (NMDAR) and nicotinic α₇ receptors [67,96]. This compound, together with L-kynurenine, is thought to be an endogenous metabolite of L-tryptophan known to block NMDAR, and it has been frequently shown to exert neuroprotective or anticonvulsant properties in the brain [33,55,62,67,96,104,145]. This compound has been disclosed to inhibit NMDARs at the glycine-binding site and it can noncompetitively inhibit α₇-nicotinic acetylcholine receptor, and through this action, it might modulate glutamate release presynaptically [62,96,97]. For instance, as administered systemically, a KYNA analog (SZR104) was shown to decrease population spike activity from the pyramidal layer of area CA1 of the hippocampus [33].

Previous studies have shown that the reduction in the astrocytic formation of KYNA could enhance glutamatergic tone in the hippocampus as well as cognitive abilities and synaptic plasticity [55,62,130]. KYNA is also recognized to be a target molecule in neuroendocrinology [130]. The KYNA derivatives have been increasing noticed to exert various biological actions [29]. Earlier work has also shown that KYNA-induced hypotension is strongly linked to the stimulation of I_K(M) magnitude [111].

Of interest, recent studies have demonstrated that GH₃-cell exposure to KYNA can result in the stimulation of I_K(M) with an EC₅₀ value of 18.1 μM. The EC₅₀ value of KYNA-stimulated I_K(M) appeared to be lower than that for its inhibition of NR1a/NR2A receptors or AMPA-evoked currents [95]. The relationship of I_K(M)-conductance versus membrane potential during cell exposure to KYNA was noted to produce a leftward shift along the voltage axis by approximately 4 mV. Therefore, it is anticipated to be a pertinent link between KYNA effects on endocrine or neuroendocrine cells and the stimulatory effect on I_K(M) magnitude.

In addition to the increased I_K(M) amplitude, the presence of KYNA can shorten the activation time constant of the current. Stimulation of I_K(M) caused by KYNA thus does not become instantaneous, yet it develops with time, once the K_M channels are opened upon membrane depolarization, thereby leading to an increase in current activation. In keeping with these observations, single-channel current recordings were found to prolong mean open time of K_M channels in the presence of KYNA. Therefore, the increase in both open-state probability and mean open time of K_M channels produced by KYNA or its amide derivatives would be responsible for its increase of macroscopic I_K(M) carried through these channels, despite their ineffectiveness in changing the single-channel amplitude. In this regard, KYNA or its structural similar compounds would be expected to be valuable tools for probing the structure and function of K_M channels [99]

D. Hyperpolarization-Activated Cation Current (I_h)

The hyperpolarization-activated cation current (I_h), also known as the “funny current” (I_f), play a crucial role in regulating repetitive electrical activity in cardiac cells, various types of central neurons, and endocrine or neuroendocrince cells [50,116,151,155,156]. This type of ionic currents exhibits unique characteristics, including slow voltage-dependent activation kinetics and a mixed Na⁺/K⁺ current that flows inwardly, and it can be blocked by CsCl or ivabradine [35,58,116]. Activation of I_h may lead to depolarization of the resting potential, reaching the threshold required for generating or triggering an AP. Consequently, it influences pacemaker activity and impulse propagation [35,133].

Additionally, the inwardly directed I_h, triggered by sustained hyperpolarization and recognized for its gradual activation, can lead to persistent, activity-dependent adjustments in membrane excitability in diverse types of excitable cells [151,155,156,157]. Furthermore, the unique voltage-dependent hysteresis of I_h evoked by double triangular ramp pulse has been demonstrated [24,91,146]. The I_h is mediated by channels encoded by members of the hyperpolarization-activated cyclic nucleotide-gated (HCN) gene family, and earlier studies have demonstrated that the activity of these channels underlies the ionic mechanisms associated with both convulsive disorders and inflammatory pain disorders [26,31,100,149,156,157].

• 1. Carisbamate (CRS, RW1-333369, Vimpat^®, (S)-2-Oxo-1-pyrrolidineacetamide)

CRS, a bioactive, orally administered neuromodulator, has been shown to be beneficial for the treatment of different types of convulsive disorders, including drug-resistant focal epilepsy and partial-onset seizure [36,88,107]. CRS is thought to work by affecting the activity of certain neurotransmitters in the brain, helping to reduce the occurrence of seizures [74]. This compound was also reported to be effective in the treatment of alcoholism [109]. Another study demonstrated that CRS prevented the development and production of epilepsy-like discharges and exerted a neuroprotective effect after epilepticus-like injury [37].

A recent study has produced an intriguing result, demonstrating that CRS can induce an inhibitory effect on I_h intrinsically in GH₃ cells, and the extent of this effect varies with the applied concentration [57]. Using the modified Hill equation, the IC₅₀ value required for CRS to suppress the I_h amplitude as seen in GH₃ cells were estimated to be 38 μM. With the use of double triangular ramp pulse, the voltage-dependent hysteresis of I_h can be robustly activated. When the GH₃ cells were continually exposed to CRS, the hysteretic strength of I_h evoked by the inverted triangular ramp pulse became progressively decreased [57]. The anticipated docking interaction between CRS and a model of the HCN channel also underscores CRS’s capacity to form hydrogen bonds and engage in hydrophobic interactions with amino acid residues in HCN channel [57]. Therefore, in addition to the inhibition of I_Na, CRS can interact directly with HCN channel to alter the magnitude, gating kinetics, and hysteretic strength of I_h present in pituitary cells. The extent to which CRS-mediated inhibition of I_h affects brain or endocrine function requires further investigation.

2

Cannabidiol (CBD, 2-[(1R,6R)-3-methyl-6-prop-1-en-2-ylcyclohex-2-en-1-yl]-5-pentylbenzene-1,3-diol)

CBD is a non-psychoactive cannabinoid derived from the Cannabis plant, known for its potential therapeutic and medicinal properties. It is among over 100 cannabinoids present in the plant and has been demonstrated to be effective at treating various medical conditions, such as epilepsy, bipolar disorder, inflammation, and cancer [14,39]. Recent work has demonstrated that CBD can modify the activity in the hypothalamic-pituitary-adrenal axis [87,131].

A current study showed the effectiveness of CBD in suppressing the magnitude of I_h in GH₃ cells and in increasing the activation time constant of the current [85]. The IC₅₀ value for CBD-mediated inhibition of I_h was calculated to be 3.3 μM, and the decrease was reversed by oxaliplatin. Oxaliplatin, a platinum-based chemotherapeutic agent, was reported to stimulate I_h [19]. The quasi-steady-state activation curve of I_h was shifted in the leftward direction with no changes in the steepness of the curve. CBD also diminished the strength of voltage-dependent hysteresis on I_h elicited by double triangular ramp pulse. Findings from recent results suggest that CBD’s modification of I_h is independent of the binding to cannabinoid or opioid receptors and may exert a significant impact on the functional activities of electrically active cells occurring in vitro or in vivo.

E. Large-conductance Ca²⁺-activated K⁺ (BK_Ca) channel

The large-conductance Ca²⁺-activated K⁺ (BK_Ca or BK) channels (KCa1.1, KCNMA1, Slo1) belong to a family of voltage-gated K⁺ channels, and they are activated by an increase in either the intracellular concentration of Ca²⁺, the membrane potential, or both. The activation of BK_Ca channels can result in conducting large amounts of K⁺ ions across the cell membrane. Due to its high-conductance state and a single-channel conductance of about 150-250 pS, the BK_Ca channel is also thought to be a maxi- or large-K⁺ channel. This family of K+ channels is functionally expressed in pituitary cells, and its activity can affect the magnitude of whole-cell Ca²⁺-activated K⁺ currents (I_K(Ca)) in pituitary cells, consequently impacting the membrane potential and stimulus-secretion coupling of these cells.

• 1. Rufinamide (RFM, Banzel^®, Inovelon^®, ethyl 1-(2,6-difluorophenyl)-1H-1,2,3-triazole-4-carboxylate)

RFM is recognized as a unique anticonvulsant drug, because, as a triazole derivative, its structure is dissimilar to other currently marked antiepileptic drugs [7,60]. It is increasingly being used in combination with other medications and therapies to treat Lennox-Gaustaut syndrome, severe epileptic encephalopathy, and other seizure disorders [10,102,112,115]. Lennox-Gaustaut syndrome is a rare and severe form of epilepsy that typically begins in childhood and it is characterized by multiple types of seizures and intellectual and developmental disabilities.

Although the mechanism of RFM action as an antiepileptic drug is still unclear, RFM was reported to modulate the activity of Na_V channels by prolonging the inactive state of these channels [124,132]. Recent studies have also shown that RFM can interact with BK_Ca channels to enhance whole-cell Ca²⁺-activated K⁺ currents (I_K(Ca)) effectively. As shown in Figure 5, the application of 10 μM RPM significantly enhances the amplitude of I_K(Ca) across the entire voltage-clamp step. The effective EC₅₀ value of RFM required for stimulating I_K(Ca) was estimated to be 3.9 μM, with a Hill coefficient of 1.2 [73]. The maximum plasma concentrations of RFM at dosages of 10 mg/kg/day and 30 mg/kg/day have been reported as 4.01 μg/ml (16.8 μM] and 8.68 μg/ml (36.4 μM], respectively [136]. Consequently, the EC₅₀ value is observed to be within the range of clinically achieved concentrations.

Additionally, the docking study showed that RFM can bind to the intracellular domain of K_Ca1.1 channel at certain amino-acid residues and that the RFM-induced docking site is not located in the pore regions of the channels [73]. Indeed, in pituitary GH₃ cells, the addition of RFM to the cytosolic surface of the detached patch of membrane resulted in the enhanced activity of BK_Ca channels, with no modification in single-channel conductance of the channel [73]. The mean closed time of BK_Ca channels was decreased by the application of RFM to the cytosolic leaflet of the channel. Overall, aside from its ability to block I_Na, similar to riluzole as demonstrated previously [138,140], RFM has been shown to effectively enhance activity of BK_Ca channels within excitable cells in in vivo settings.

• 2 QO-40 ((5-(chloromethyl)-3-(naphthalen-1-yl)-2-(trifluoromethyl)pyrazolo [1,5-a]pyrimidin-7(4 H)-one)

QO-40 is a highly pure, synthetic, and biologically active compound. This compound has been previously reported to enhance KCNQ2/KCNQ3 heteromeric currents expressed in Xenopus oocytes [61]. QO58-lysine, a compound structurally similar to QO-40, can also activate neuronal KCNQ channels and exert antinociceptive effects on inflammatory pain [127]. The QO-58-induced amelioration of inflammatory pain observed in rodents was previously viewed as being accompanied by the activation of KCNQ-encoded K⁺ currents [127,152].

In a recent study [22], as pituitary GH₃ cells were exposed to QO-40, the magnitude of I_K(Ca) was observed to be notably increased with an EC₅₀ value of 2.3 μM. QO-40-stimulated I_K(Ca) was attenuated by further addition of paxilline, yet not by linopirdine or TRAM-34. It is worth noting that paxilline is a tremorgenic mycotoxin known to suppress the activity of BK_Ca channels [68], while linopirdine inhibits the I_K(M) magnitude, and TRAM-34 can suppress the activity of intermediate-conductance Ca²⁺-activated K⁺ channels [147]. In inside-out single-channel recordings, it was observed that QO-40 not only produced a 14 mV shift towards a less positive potential in the steady-state activation curve of BK_Ca channels but also increased the gating charge by 1.4-fold. However, it is important to highlight that QO-40 did not alter the single-channel conductance of the channel, despite causing a reduction in the mean closed time of BK_Ca channels when it was present. Additionally, with the long-lasting isosceles-triangular ramp pulse, the presence of QO-40 enhanced the voltage-dependent hysteretic strength of BK_Ca channels. Although the detailed mechanism of the stimulatory actions of QO-40 on BK_Ca channels is not yet known, experimental observations suggest that QO-40 can enhance the activity of BK_Ca channels in a voltage-dependent manner [22]. As a result, its interaction with the BK_Ca channel can vary significantly based on factors such as the resting potential, AP firing pattern, the concentration of QO-40 used, or any combination of these variables.

The maximal concentration of QO-58, a synthesized compound that is structurally similar to QO-40, following oral administration at 25, 50, or 100 mg/kg, has been reported to reach 8.25, 16.29, or 18.27 mg/liter (approximately 18.6, 37, or 41 μM), respectively [84]. In this scenario, the stimulatory effect of QO-40 on BK_Ca channels would be of pharmacological or therapeutic relevance, as this compound at lower concentrations is effective at stimulating I_K(Ca) and enhancing BK_Ca channel activity. However, it remains to be answered whether the rank order for QO-40 or other chemically related agents in activating BK_Ca channels would share a similar magnitude for their stimulation of neuronal KCNQ currents.

2. Conclusions

This paper does not focus voltage-independent currents, such as those mediated by transient receptor potential (TRP) channels, including TRPC, TRPM, and TRPV [65]. However, it provides a proof-of-concept for understanding the pathophysiological and pharmacological roles of pituitary cells, with a particular emphasis on ion channel functionality. This review also examines how specific drugs or compounds influence the intrinsic voltage-gated ionic currents within pituitary cells. These insights are crucial for regulating the function of electrically active cells and advancing our understanding of pituitary neuroendocrine tumors (PitNets). Notably, rats can also develop PitNets, or pituitary tumors, in their pituitary glands, much like humans and some other animals.

It is important to note that the current pituitary cell lines primarily used in research are derived from rats, such as GH₃ cells, GH₄C₁ cells, MMQ, R1220, and AtT-20 cells [22,49,54,76,83,119,158]. Detailed descriptions of these cell lines can be found in Table 2. However, there are limited reports on immortalized human pituitary cell lines. Whether the electrical properties observed in these cell lines, as well as their responses to various drugs, are preserved in human pituitary cells remains an area that requires further in-depth investigation. In addition to pituitary cells, other endocrine cell types―such as those responsible for insulin and glucagon secretion, as well as Leydig cells―have also been shown to exhibit similar ionic currents [8,25,28,47,71]. However, further research is needed to determine whether the drugs or compounds mentioned here impact the electrical activity of these different endocrine cell types and their functional implications. Understanding the regulation of ion currents discussed in this paper is crucial for unraveling the molecular mechanisms underlying PiNets and their potential for advancing therapeutic approaches [23,159,160,161].

In clinical practice, it is common to observe that many patients with PitNets develop tumors in other endocrine glands, leading to an occurrence of an endocrine tumor syndrome known as multiple endocrine neoplasia type 1 (Wermer syndrome) [6,34,45,106,159,160]. Computerized tomography-guided radiofrequency ablation has become a key treatment option for removing PiNets removal, helping to minimize the risk of damage to surrounding deep brain tissues [162]. Additionally, the development of different medications aimed at preventing the recurrence of such tumors or other forms of multiple endocrine neoplasia will be an important topic for future research. At the same time, it is also an important task to conduct in-depth investigations of potential genetic abnormalities in patient samples of these PiNets [163]. Therefore, the comprehensive research presented in this paper will play a crucial role in advancing our comprehension of the origin and management of these conditions.

Although techniques like polymerase chain reaction (PCR) and Western blotting can be used to measure gene and protein expression abnormalities, respectively, in PitNets, performing patch-clamp experiments on different pituitary cells enables direct investigation of the biophysical properties of individual or whole ionic currents across the cell membrane, as well as changes in membrane potential, as presented herein. Therefore, it remains a crucial approach for functional studies. Additionally, performing direct measurements on PitNets’ organoid (165) will open up further opportunities for detailed pituitary research in the future.