Ca2+ Signaling and Cell Death Induced by Protriptyline in HepG2 Human Hepatoma Cells
Abstract
The physiological mechanisms by which protriptyline might influence cellular processes, particularly calcium ion dynamics within human hepatoma cells, have remained largely unexplored. This investigation was meticulously designed to elucidate the precise effects of protriptyline on intracellular calcium concentrations, denoted as [Ca2+]i, and to assess its cytotoxic potential within the well-established HepG2 human hepatoma cell line. The findings revealed that the administration of protriptyline, across a concentration range of 50 to 150 micromolar, consistently elicited significant elevations in [Ca2+]i. A critical observation was that the influx of extracellular Ca2+ appeared to be a primary contributor to this increase, as the removal of Ca2+ from the external medium substantially diminished the protriptyline-induced calcium rise. Further corroboration of this extracellular calcium entry mechanism was achieved through the observation of manganese ion (Mn2+)-induced quenching of fura-2 fluorescence, a widely accepted technique for monitoring calcium influx pathways.
Investigating the specific channels and pathways involved in this calcium entry, it was determined that nifedipine, a known antagonist of certain voltage-gated calcium channels, effectively inhibited the protriptyline-induced Ca2+ entry. Conversely, several other compounds, including econazole, SKF96365, GF109203X, and phorbol 12-myristate 13 acetate, did not exhibit similar inhibitory effects on the observed Ca2+ entry, suggesting a specific interaction with nifedipine-sensitive channels. Furthermore, the study explored the contribution of intracellular calcium stores to the overall response. Pre-treatment with 2,5-di-tert-butylhydroquinone (BHQ), a potent inhibitor of the endoplasmic reticulum Ca2+ pump, was found to suppress approximately 40% of the protriptyline-induced [Ca2+]i response, indicating a significant role for endoplasmic reticulum calcium release. Intriguingly, subsequent exposure to protriptyline completely abolished any further BHQ-induced calcium response, suggesting that protriptyline might deplete these internal stores or interfere with their refilling. The involvement of the crucial signaling enzyme phospholipase C (PLC) was also examined, with its inhibition leading to a substantial 70% suppression of the protriptyline-evoked [Ca2+]i rise, thereby highlighting a major PLC-dependent component in the overall calcium signaling pathway activated by protriptyline. Beyond its effects on calcium dynamics, protriptyline also demonstrated a dose-dependent cytotoxicity. At concentrations ranging from 20 to 40 micromolar, protriptyline effectively induced cell death in HepG2 cells. Notably, this cytotoxic effect could not be reversed or mitigated by the pre-treatment with 1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid-acetoxymethyl ester (BAPTA/AM), a highly effective intracellular Ca2+ chelator. This latter finding strongly suggests that the mechanism of cell death induced by protriptyline in HepG2 cells operates independently of intracellular calcium elevation. Collectively, these comprehensive findings establish that in HepG2 cells, protriptyline provokes rises in [Ca2+]i through a dual mechanism: both via calcium entry through nifedipine-sensitive calcium channels and through a significant phospholipase C-dependent release of calcium from the endoplasmic reticulum. Furthermore, protriptyline uniquely induces cell death in these hepatoma cells through a pathway that is demonstrably independent of calcium signaling.
Introduction
For several decades, protriptyline has been recognized and clinically utilized primarily as an antidepressant, contributing significantly to the management of mood disorders. Its therapeutic utility, however, extends beyond its initial application. Historical clinical reports have documented its diverse actions, encompassing the alleviation of symptoms associated with conditions such as tinnitus, certain forms of respiratory failure, and obstructive sleep apnea. Furthermore, its application has been explored in the context of obstructive pulmonary disease, snoring, the sequelae of brain injury, and even hypoxemia, showcasing its broad pharmacological profile. The compound has also been investigated for its potential in addressing attention-deficit hyperactivity disorder, demonstrating a wide range of documented clinical efficacy.
Complementary to its clinical applications, considerable effort has been dedicated to unraveling the fundamental in vitro effects of protriptyline across various cell types, aiming to delineate its cellular mechanisms of action. Prior research, for instance, has demonstrated protriptyline’s capacity to modulate ion channel activity, specifically its ability to suppress potassium channels within isolated rat sympathetic neurons. In a related vein, other studies have reported that protriptyline effectively blocks the human ether-a-go-go-related gene (hERG) potassium channel, a crucial channel involved in cardiac repolarization. Beyond ion channel modulation, evidence has also emerged suggesting that protriptyline can induce DNA damage, highlighting its potential impact on genomic integrity. Despite these important insights into its various cellular interactions, a significant void persists in our understanding of protriptyline’s specific effects on calcium ion signaling within human hepatoma cells. This lacuna in knowledge represents a critical area for investigation, given the central role of calcium in cellular physiology and pathophysiology.
Changes in intracellular free calcium concentrations, [Ca2+]i, serve as a fundamental and ubiquitous messenger, orchestrating a vast array of cellular processes and modulating diverse cellular responses. Calcium signaling is intimately involved in functions ranging from gene expression and cell proliferation to apoptosis and cell differentiation. Given its pivotal role, cells have evolved intricate and highly regulated mechanisms to precisely control [Ca2+]i. This tight regulation is essential for maintaining cellular homeostasis and for ensuring the fidelity of calcium signaling pathways. Among the sophisticated mechanisms governing calcium dynamics are various members of the superfamily of G-protein-coupled receptors (GPCRs), which are characterized by their distinctive seven transmembrane domains. Activation of these receptors frequently leads to the stimulation of phospholipase C (PLC), an enzyme that plays a central role in generating inositol trisphosphate (IP3) and diacylglycerol (DAG). The production of IP3, in particular, triggers the release of Ca2+ from intracellular stores, predominantly the endoplasmic reticulum, which in turn can lead to a subsequent influx of Ca2+ across the plasma membrane from the extracellular environment. Consequently, a detailed exploration of the precise mechanisms by which a chemical compound induces alterations in [Ca2+]i is of paramount importance. Such investigations are crucial for gaining a comprehensive understanding of the compound’s pharmacological effects on cellular function and for potentially identifying novel therapeutic targets.
Considering the existing knowledge gap, the specific objective of the present study was to systematically investigate the effect of protriptyline on intracellular calcium concentrations in HepG2 human hepatoma cells. This cell line, owing to its well-characterized properties and genetic stability, represents an invaluable and widely utilized model system for hepatoma research. Previous studies utilizing HepG2 cells have successfully demonstrated that various agents, such as tamoxifen and polyphyllin D, can induce significant [Ca2+]i rises and ultimately lead to cell death within this model system. However, these earlier investigations did not delve into the specific upstream and downstream signaling pathways that underpin these observed calcium signals. Given that even subtle alterations in [Ca2+]i can profoundly impact numerous cellular processes, it becomes imperative to meticulously explore the precise pathways responsible for protriptyline-induced [Ca2+]i elevations. Such an in-depth mechanistic understanding is indispensable for thoroughly deciphering the pharmacological effects of protriptyline on hepatoma cells and for potentially identifying its suitability as a therapeutic agent. To achieve these aims, the study employed fura-2, a highly sensitive fluorescent calcium-indicator dye, to precisely measure changes in [Ca2+]i. A comprehensive characterization of protriptyline-induced [Ca2+]i rises was performed, including the establishment of detailed concentration-response plots. Crucially, the investigation also focused on dissecting the specific signaling pathways underlying these calcium elevations. Furthermore, an assessment of protriptyline’s impact on cell viability was meticulously conducted to determine any associated cytotoxic effects.
Materials And Methods
Materials
The comprehensive range of reagents necessary for routine cell culture procedures was procured from Gibco, a well-established supplier in Gaithersburg, MD, ensuring consistency and quality. Aminopolycarboxylic acid/acetoxy methyl, commonly known as fura-2/AM, which serves as a pivotal fluorescent indicator for intracellular calcium, and 1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid/acetoxy methyl, or BAPTA/AM, a widely used intracellular calcium chelator, were both obtained from Molecular Probes located in Eugene, OR. These specialized reagents are critical for accurate calcium measurements and for investigating calcium-dependent cellular processes. All other chemical reagents and compounds utilized throughout the experimental procedures, unless specifically indicated otherwise, were sourced from Sigma–Aldrich in St. Louis, MO, upholding a standard of purity and reliability in the preparation of solutions and experimental media.
Cell Culture
The HepG2 human hepatoma cells, which served as the experimental model for this study, were acquired from the Bioresource Collection and Research Center situated in Taipei, Taiwan, ensuring their authenticated origin and genetic integrity. These cells were meticulously maintained and cultured in a highly optimized Minimum Essential Medium. This basal medium was appropriately supplemented with 10% heat-inactivated fetal bovine serum, which provides essential growth factors and nutrients, along with a protective combination of 100 U/mL penicillin and 100 µg/mL streptomycin, acting as broad-spectrum antibiotics to prevent microbial contamination during long-term culture. The cultivation conditions were carefully controlled to promote optimal cell proliferation and viability, ensuring the reliability of subsequent experimental measurements.
Solutions Used in [Ca2+]i Measurements
For the precise measurement of intracellular calcium concentrations, specific buffered solutions were meticulously prepared. The standard Ca2+-containing medium was formulated to mimic physiological extracellular conditions, with a carefully maintained pH of 7.4. Its composition included 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2 (as the primary source of extracellular calcium), 10 mM HEPES (a zwitterionic buffering agent crucial for maintaining pH stability), and 5 mM glucose (to provide an energy source for cellular metabolism). In experiments requiring the absence of extracellular calcium, a Ca2+-free medium was prepared. This solution contained similar chemical constituents to the Ca2+-containing medium, with the critical modifications being the complete omission of CaCl2 and its replacement with 0.3 mM EGTA, a calcium-chelating agent, to effectively bind any residual calcium ions. Additionally, 2 mM MgCl2 was included in the Ca2+-free medium. Protriptyline, the compound under investigation, was initially dissolved in ethanol to create a highly concentrated 0.1 M stock solution, allowing for precise dilution into experimental media. Other chemical reagents used in the study were dissolved in appropriate solvents, including water, ethanol, or dimethyl sulfoxide (DMSO), depending on their solubility characteristics. A stringent control was maintained over the concentration of organic solvents in the final experimental solutions; their presence never exceeded 0.1%. Rigorous preliminary experiments confirmed that at this low concentration, the organic solvents had no discernible effect on basal [Ca2+]i levels or on cell viability, thereby ensuring that any observed effects were solely attributable to protriptyline.
[Ca2+]i Measurements
The methodology for measuring intracellular free calcium concentrations ([Ca2+]i) was rigorously followed as previously established and described. To prepare for measurements, HepG2 cells, once they had reached confluence on 6 cm culture dishes, were carefully trypsinized to detach them from the substrate. The detached cells were then gently suspended in fresh culture medium to achieve a uniform concentration of 10^6 cells/mL. Prior to calcium loading, the viability of the cell suspension was assessed using the trypan blue exclusion method. This critical quality control step confirmed that cell viability consistently remained above 95% compared to the control untreated cells, ensuring that only healthy and intact cells were used for subsequent analyses. Subsequently, the cells were incubated with 2 µM fura-2/AM, a cell-permeant derivative of the calcium indicator fura-2, for a period of 30 minutes at 25°C in the same culture medium. This incubation allowed for the intracellular esterases to cleave the AM group, trapping the active fura-2 dye within the cytoplasm. Following the loading period, cells were washed twice with Ca2+-containing medium to remove any residual extracellular fura-2/AM. Finally, the cells were resuspended in Ca2+-containing medium at a higher concentration of 10^7 cells/mL, optimizing them for fluorescence measurements.
Fura-2 fluorescence measurements were conducted in a specialized water-jacketed cuvette, maintaining a constant temperature of 25°C with continuous stirring to ensure uniform distribution of cells and reagents. Each measurement was performed in a cuvette containing 1 mL of medium and approximately 0.5 million cells. Fluorescence was continuously monitored using a Shimadzu RF-5301PC spectrofluorophotometer, a highly sensitive instrument capable of detecting subtle changes in fluorescence intensity. Immediately after the addition of 0.1 mL of cell suspension to 0.9 mL of either Ca2+-containing or Ca2+-free medium in the cuvette, recordings commenced. The system was set to record excitation signals alternately at 340 nm and 380 nm, while monitoring the emission signal at 510 nm, at precise 1-second intervals. During the recording sessions, reagents of interest were introduced into the cuvette by briefly pausing the recording for 2 seconds to allow for the opening and closing of the cuvette chamber, ensuring minimal disruption to the measurement kinetics.
For the accurate calibration of absolute [Ca2+]i values, a standard procedure was followed upon the completion of each experiment. First, the non-ionic detergent Triton X-100 (0.1%) was added to permeabilize the cell membranes, followed by the addition of 5 mM CaCl2 to saturate all intracellular and extracellular fura-2 with calcium, thereby obtaining the maximal fura-2 fluorescence signal. Subsequently, 10 mM EGTA, a high-affinity Ca2+ chelator, was introduced to bind all available calcium in the cuvette, yielding the minimal fura-2 fluorescence signal. These maximal and minimal fluorescence values are essential for calculating [Ca2+]i using a standard formula that corrects for dye properties and background fluorescence. Control experiments confirmed the robustness of the experimental setup, showing that cells maintained a viability of approximately 95% even after 20 minutes of continuous fluorescence measurements within the cuvette, assuring the physiological integrity of the cells throughout the duration of the experiment. Intracellular calcium concentrations were calculated precisely using a previously established and validated method, ensuring accuracy and comparability of results.
To specifically confirm extracellular Ca2+ entry, the Mn2+ quenching of fura-2 fluorescence technique was employed. This method leverages the fact that Mn2+ ions, when entering the cell through plasma membrane Ca2+ channels, can bind to fura-2 and quench its fluorescence signal at the isosbestic point (360 nm), where fura-2 fluorescence is independent of Ca2+ concentration. The experiments were performed in Ca2+-containing medium supplemented with 50 µM MnCl2. MnCl2 was introduced into the cell suspension within the cuvette 30 seconds prior to the initiation of fluorescence recording. The data were collected by monitoring the excitation signal at 360 nm and the emission signal at 510 nm at 1-second intervals, as meticulously described in prior literature, providing a direct optical measure of transmembrane metal ion flux.
Cell Viability Assays
Cell viability was rigorously assessed following a previously established and validated protocol. The fundamental principle underlying the measurement of cell viability in this study relied on the metabolic activity of living cells, specifically their capacity to cleave tetrazolium salts through the action of mitochondrial dehydrogenases. This enzymatic reaction produces a colored formazan product; consequently, an increase in the intensity of the color directly correlates with the number of viable and metabolically active cells. The assays were conducted strictly in accordance with the manufacturer’s detailed instructions provided by Roche Molecular Biochemical (Indianapolis, IN), ensuring the standardization and reproducibility of the results.
For the viability assessment, HepG2 cells were initially seeded into 96-well plates at a standardized density of 10,000 cells per well. They were then allowed to incubate in complete culture medium for a period of 24 hours. Following this initial incubation, the cells were exposed to varying concentrations of protriptyline, ranging from 0 to 40 micromolar, for an additional 24 hours. After the protriptyline treatment, a cell viability detecting tetrazolium reagent, specifically 4-[3-[4-Iodophenyl]-2-4(4-nitrophenyl)-2H-5-tetrazolio-1,3-benzene disulfonate] (WST-1), prepared as a 10 micromolar pure solution, was carefully added to each sample well. The cells were then further incubated for 30 minutes in a humidified atmosphere, allowing sufficient time for the enzymatic conversion to occur.
In specific experiments designed to investigate the role of cytosolic Ca2+ in protriptyline-induced cell death, cells were pre-treated with 5 µM BAPTA/AM, the intracellular Ca2+ chelator, for one hour prior to the incubation with protriptyline. This pre-treatment ensures that intracellular Ca2+ is effectively buffered, thereby allowing for the determination of whether the cytotoxic effects are Ca2+-dependent. Following the BAPTA/AM pre-treatment, the cells were gently washed once with Ca2+-containing medium to remove any residual extracellular chelator before being subsequently incubated with or without protriptyline for 24 hours. The absorbance of the treated samples was determined at 450 nm (A450) using an ELISA reader. This optical density measurement provides a quantitative readout of the formazan product. To facilitate comparative analysis and to account for any plate-to-plate variability, the absolute optical density values obtained from each sample were meticulously normalized to the absorbance of unstimulated control cells within each respective plate. The results were then expressed as a percentage of the control value, providing a clear and standardized measure of cell viability relative to untreated conditions.
Statistics
All experimental data presented in this study are meticulously reported as the mean value plus or minus the standard error of the mean (SEM), derived from a minimum of three independent experimental replicates. For statistical analysis, the data were rigorously subjected to analysis of variance (ANOVA) utilizing the Statistical Analysis System (SAS), specifically SAS Institute Inc., located in Cary, NC. To identify statistically significant differences between multiple group means, a post-hoc analysis was subsequently performed. The Tukey’s HSD (honestly significant difference) procedure was employed for these multiple comparisons, a conservative and robust method that minimizes the risk of Type I errors when numerous comparisons are made. A P value of less than 0.05 was prospectively established as the threshold for statistical significance, indicating that observed differences were unlikely to have occurred by chance.
Results
Effect of Protriptyline on [Ca2+]i
The initial phase of this investigation systematically examined the influence of protriptyline on the basal intracellular calcium concentration ([Ca2+]i) in HepG2 cells. The resting [Ca2+]i level was consistently measured at 51 ± 2 nM, establishing a baseline for subsequent experimental observations. Introduction of protriptyline, within a concentration range spanning from 50 to 150 micromolar, reliably induced a discernible increase in [Ca2+]i. This elevation exhibited a clear concentration-dependent relationship when the experiments were conducted in a Ca2+-containing medium, indicating that higher concentrations of protriptyline elicited more pronounced calcium responses. Specifically, at a concentration of 150 micromolar, protriptyline provoked a robust [Ca2+]i rise that achieved a net increase of 333 ± 6 nM (n = 3), followed by a sustained and prolonged plateau phase, suggesting a maintained cellular response. Further increasing the protriptyline concentration to 200 micromolar did not yield a significantly greater calcium response, indicating that the Ca2+ response had reached saturation at the 150 micromolar concentration.
To differentiate between calcium influx from the extracellular environment and release from intracellular stores, experiments were also conducted in a Ca2+-free medium. Under these conditions, protriptyline, again at concentrations ranging from 50 to 150 micromolar, continued to induce concentration-dependent rises in [Ca2+]i, albeit with a different kinetic profile. This observation confirms the involvement of intracellular calcium stores in the protriptyline-induced response. Comprehensive concentration-response plots were constructed, illustrating the relationship between protriptyline concentration and the magnitude of the [Ca2+]i rises. Analysis of these plots, by fitting them to a Hill equation, yielded an EC50 value of 100 ± 3 micromolar when experiments were performed in Ca2+-containing medium, and a very similar EC50 value of 102 ± 2 micromolar in Ca2+-free medium. The proximity of these EC50 values further underscores the dual contribution of both extracellular calcium entry and intracellular calcium release to the overall protriptyline-induced calcium signal.
Protriptyline-Induced Mn2+ Influx
To definitively ascertain whether the protriptyline-evoked [Ca2+]i rises involved an influx of extracellular calcium ions, a series of experiments employing the manganese (Mn2+) quenching of fura-2 fluorescence technique were performed. This method is based on the principle that Mn2+ ions, which enter cells through similar plasma membrane channels as Ca2+, have the unique property of quenching fura-2 fluorescence across all excitation wavelengths, including the Ca2+-insensitive wavelength of 360 nm. Therefore, a measurable decrease in fura-2 fluorescence excited at 360 nm in the presence of Mn2+ serves as a direct indicator of transmembrane Mn2+ entry, and by extension, Ca2+ influx. Given that the protriptyline-induced calcium response had been determined to saturate at 150 micromolar, this concentration was consistently used as the reference point for subsequent experiments. The results clearly demonstrated that exposure to 150 micromolar protriptyline elicited an immediate and sustained decrease in the 360 nm excitation signal, which reached a notable value of 62 ± 4 arbitrary units by the 250-second mark. This significant quenching of fura-2 fluorescence by Mn2+ unequivocally suggests that extracellular calcium influx is a substantial component contributing to the overall protriptyline-evoked increases in [Ca2+]i.
Nifedipine was Involved in Protriptyline-Induced Ca2+ Entry
To meticulously investigate the specific pathways mediating the extracellular Ca2+ entry triggered by protriptyline, a series of experiments were undertaken utilizing various pharmacological modulators of calcium channels. The agents tested included nifedipine, a known antagonist of L-type voltage-gated calcium channels; econazole (at 0.5 µM) and SKF96365 (at 5 µM), both recognized inhibitors of store-operated Ca2+ entry (SOCE); phorbol 12-myristate 13 acetate (PMA; at 1 nM), an activator of protein kinase C (PKC); and GF109203X (at 2 µM), a selective PKC inhibitor. Each of these modulators was applied to the cells one minute prior to the addition of 150 micromolar protriptyline, allowing sufficient time for their respective inhibitory or activating effects to take hold.
The findings revealed that PMA, GF109203X, econazole, and SKF96365 all failed to exert any significant inhibitory effect on the protriptyline-induced [Ca2+]i rises, suggesting that PKC modulation and typical store-operated calcium channels are not the primary pathways for protriptyline-mediated calcium entry in HepG2 cells. In stark contrast, nifedipine proved to be a notable exception, effectively inhibiting the protriptyline-induced [Ca2+]i rise by a significant margin of 33 ± 2%. This specific inhibition by nifedipine strongly implicates the involvement of nifedipine-sensitive calcium channels, which are typically associated with voltage-gated calcium channels, in mediating a portion of the protriptyline-induced Ca2+ influx. This suggests a distinct mechanism of entry compared to the canonical store-operated pathways.
Sources of Protriptyline-Induced Ca2+ Release
In the vast majority of cell types, including the HepG2 cells utilized in this study, the endoplasmic reticulum (ER) stands out as the predominant intracellular calcium storage organelle, playing a central role in calcium homeostasis and signaling. Consequently, a dedicated series of experiments was designed to specifically elucidate the contribution of the endoplasmic reticulum to the protriptyline-evoked calcium release in HepG2 cells. To isolate the effects of intracellular calcium release from those of extracellular calcium influx, these experiments were strictly performed in a Ca2+-free medium.
In the first experimental setup, after the HepG2 cells had responded to 150 micromolar protriptyline with an initial [Ca2+]i rise, the addition of 50 micromolar 2,5-di-tert-butylhydroquinone (BHQ), a well-established inhibitor of the endoplasmic reticulum Ca2+ pump (SERCA), failed to induce any further discernible [Ca2+]i rises. This suggests that protriptyline might deplete the BHQ-sensitive ER calcium stores or interfere with their ability to reaccumulate calcium. Conversely, in a different sequence, when BHQ was introduced first, it successfully elicited a [Ca2+]i rise of 45 ± 2 nM, indicative of its capacity to release calcium from the ER stores by inhibiting the pump that reuptakes calcium. Subsequent addition of 150 micromolar protriptyline to these BHQ-pre-treated cells still induced a further [Ca2+]i rise, but this response was approximately 40% smaller in terms of the area under the curve compared to the response observed when protriptyline was added alone without prior BHQ treatment. This reduction strongly implies that a significant portion of the protriptyline-induced calcium release originates from BHQ-sensitive endoplasmic reticulum stores.
Given that protriptyline demonstrably released calcium from the endoplasmic reticulum, the investigation proceeded to examine the involvement of phospholipase C (PLC), a key enzyme known to regulate the release of Ca2+ from intracellular stores in many cellular systems. To ascertain if the activation of PLC was an essential prerequisite for protriptyline-evoked Ca2+ release, the PLC inhibitor U73122 was employed. It is widely understood that activation of PLC typically leads to the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). While DAG is known to activate protein kinase C (PKC), IP3 is the critical messenger that binds to its specific receptors on the endoplasmic reticulum, thereby triggering the release of stored calcium.
Prior to testing protriptyline, the efficacy of U73122 was validated using ATP, a well-known PLC-dependent agonist of [Ca2+]i rises in various cell types. The results confirmed that 10 micromolar ATP reliably induced [Ca2+]i rises of 48 ± 3 nM. Crucially, pre-incubation with 2 micromolar U73122 did not alter the basal [Ca2+]i but completely abolished the ATP-induced [Ca2+]i rises, unequivocally demonstrating that U73122 effectively suppressed PLC activity in these cells. Having validated its effectiveness, U73122 was then applied to investigate the protriptyline response. The data showed that incubation with 2 micromolar U73122, while not affecting basal [Ca2+]i, significantly inhibited approximately 70 ± 2% of the 150 micromolar protriptyline-induced [Ca2+]i rises. This substantial inhibition strongly implicates a major PLC-dependent mechanism in the protriptyline-evoked calcium release from intracellular stores. As a crucial control, U73343 (at 2 µM), a structural analogue of U73122 that lacks PLC inhibitory activity, was also tested and, as expected, did not exert any inhibitory effect on the protriptyline-induced calcium response, further supporting the specificity of U73122′s action on PLC.
Effect of Protriptyline on Cell Viability
Considering that acute exposure to protriptyline elicited substantial elevations in intracellular calcium concentrations, and recognizing the well-established principle that dysregulated or excessive [Ca2+]i rises can frequently lead to alterations in cell viability, a subsequent series of experiments was undertaken to comprehensively assess the long-term effect of protriptyline on the viability of HepG2 cells. For this assessment, HepG2 cells were exposed to a range of protriptyline concentrations, from 0 to 40 micromolar, for an extended period of 24 hours. Following this incubation, the tetrazolium assay, a colorimetric method sensitive to metabolic activity, was performed to quantify cell viability. The results clearly indicated a dose-dependent decrease in cell viability when cells were treated with protriptyline at concentrations between 20 and 40 micromolar. This finding suggests that protriptyline, within this concentration range, exhibits a significant cytotoxic effect on HepG2 hepatoma cells over a 24-hour period.
Lack of Effect of BAPTA/AM on Reversing Protriptyline-Induced Cell Death
A critical question arising from the observed cytotoxicity was whether the protriptyline-induced cell death was a direct consequence of the preceding increases in intracellular calcium concentrations. To address this, the intracellular Ca2+ chelator BAPTA/AM, a compound known for its ability to effectively buffer and prevent sustained rises in cytosolic Ca2+, was utilized during the protriptyline treatment. Preliminary experiments confirmed the efficacy of BAPTA/AM: after pre-treatment with 5 micromolar BAPTA/AM, 150 micromolar protriptyline failed to evoke any measurable [Ca2+]i rises, thereby demonstrating that BAPTA/AM successfully chelated cytosolic Ca2+. Subsequently, the impact of BAPTA/AM on cell viability was examined. The data showed that loading cells with 5 micromolar BAPTA/AM by itself did not alter the baseline cell viability in control groups, confirming its non-toxic nature at the concentration used. Importantly, when HepG2 cells were exposed to protriptyline at concentrations of 20 to 40 micromolar in the presence of BAPTA/AM, there was no significant reversal of the protriptyline-induced cell death (n = 3; P > 0.05). This compelling finding indicates that the cytotoxic effects of protriptyline in HepG2 cells are largely dissociated from, and not directly mediated by, the increases in intracellular calcium concentrations. Despite protriptyline’s ability to elevate [Ca2+]i, the mechanism leading to cell death appears to be calcium-independent.
Discussion
Previous research has provided substantial evidence indicating that protriptyline exerts modulatory effects on ion channels in various cell types, specifically demonstrating its ability to influence potassium channels in isolated rat sympathetic neurons and in human embryonic kidney 293 cells. Building upon this existing knowledge, the current comprehensive study expands our understanding by unequivocally demonstrating that protriptyline elicits significant rises in intracellular calcium concentration ([Ca2+]i) within HepG2 human hepatoma cells. Our findings indicate that protriptyline, at concentrations ranging from 50 to 150 micromolar, induces these [Ca2+]i elevations through a dual mechanism: both by triggering the release of calcium from internal cellular stores and by promoting the influx of extracellular calcium. This conclusion is strongly supported by the observation that the removal of extracellular Ca2+ from the medium substantially diminished the protriptyline-induced calcium responses throughout the entire measurement period, directly implicating a continuous calcium influx during the cellular response. Further robust confirmation of this extracellular calcium entry was provided by the manganese (Mn2+) quenching data, which showed a clear and immediate decrease in fura-2 fluorescence, a direct optical signature of Mn2+ influx, thus paralleling the Ca2+ influx pathway.
A critical aspect of calcium signaling in HepG2 cells, as documented in numerous prior studies, centers around store-operated Ca2+ channels, which are typically activated upon depletion of intracellular calcium stores. However, our investigation suggests a more nuanced mechanism for protriptyline-induced Ca2+ entry. The data indicate that protriptyline appears to primarily cause Ca2+ entry via stimulating nifedipine-sensitive, non-store-operated Ca2+ entry pathways. This distinction is crucial, as nifedipine, a known blocker of voltage-gated calcium channels, effectively inhibited a significant portion of the protriptyline-induced [Ca2+]i rises, whereas econazole and SKF96365, which are widely recognized inhibitors of store-operated Ca2+ entry, failed to exert similar inhibitory effects. This suggests that the calcium influx pathways engaged by protriptyline in HepG2 cells differ from the canonical store-operated calcium entry mechanisms. This observation is particularly noteworthy when contrasted with findings in other cell types, such as PC3 prostate cancer cells, where protriptyline has been shown to induce Ca2+ influx primarily through store-operated Ca2+ channels. Such discrepancies highlight that the precise pathways governing protriptyline-induced calcium movement can vary significantly depending on the specific cellular context and cell type under investigation.
Furthermore, the study explored the potential involvement of protein kinase C (PKC) in the protriptyline-induced calcium signal, given the known association of PKC with calcium homeostasis. However, our experiments, which involved both activation and inhibition of PKC, revealed that neither modulating PKC activity significantly altered the protriptyline-evoked [Ca2+]i responses. This suggests that, in HepG2 cells, PKC does not play a substantial role in mediating or contributing to the protriptyline-induced calcium signal. This finding further distinguishes the mechanism in HepG2 cells from that reported in PC3 cells, where protriptyline was shown to induce PKC-sensitive Ca2+ influx, reinforcing the notion that different cell types may indeed exhibit distinct mechanisms of calcium signaling in response to the same compound.
Regarding the specific intracellular calcium stores that contribute to protriptyline-evoked Ca2+ release, our findings strongly implicate the 2,5-di-tert-butylhydroquinone (BHQ)-sensitive endoplasmic reticulum stores. Pre-treatment with BHQ significantly reduced the protriptyline-induced calcium response, indicating a substantial contribution from the ER. However, since BHQ did not completely abolish the protriptyline-induced Ca2+ release, it is plausible that protriptyline might also mobilize calcium from other, perhaps minor, intracellular stores that are not sensitive to BHQ. Crucially, the data unequivocally demonstrate that the majority of this intracellular calcium release occurs via a phospholipase C (PLC)-dependent mechanism. This conclusion is supported by the observation that when PLC activity was suppressed, the protriptyline-induced calcium release was inhibited by a remarkable 70%. The remaining 30% of the calcium release, which was independent of PLC inhibition, suggests the potential involvement of alternative, as-yet-unidentified mechanisms, which could include pathways such as those mediated by phospholipase A2 or NADPH oxidase, underscoring the complexity of intracellular calcium regulation.
Beyond its effects on calcium dynamics, the study also investigated the cytotoxic potential of protriptyline. Consistent with previous reports in other cell models, such as rat isolated sympathetic neurons and PC3 cells, protriptyline demonstrated significant cytotoxicity to HepG2 cells at concentrations ranging from 20 to 40 micromolar. In HepG2 cells, protriptyline consistently induced concentration-dependent rises in [Ca2+]i within the range of 50–150 micromolar. While it is well-established that excessive intracellular calcium overloading can profoundly alter cell viability and lead to cell death, our findings present a compelling dissociation between the protriptyline-induced cell death and the observed [Ca2+]i rises. This crucial distinction is evidenced by the fact that the cytotoxic effects of protriptyline could not be reversed or mitigated by the effective chelation of cytosolic Ca2+ with BAPTA/AM. This indicates that despite its ability to elevate intracellular calcium, the ultimate pathway leading to HepG2 cell death induced by protriptyline operates independently of these calcium fluctuations. Nevertheless, it is important to acknowledge that even if not directly causal for cell death, the increased [Ca2+]i could still significantly affect numerous other Ca2+-dependent processes within the cell, thereby altering overall cellular physiology in ways that warrant further investigation.
From a clinical perspective, it is relevant to note that plasma concentrations of protriptyline in patients can reach approximately 10 micromolar. This level can potentially be higher in individuals with underlying liver or kidney disorders, or in those receiving higher therapeutic doses. For instance, in patients with depression, the plasma concentration of protriptyline after oral administration has been reported to be up to threefold higher than in healthy adults. Given that our data demonstrate protriptyline-induced cytotoxicity at concentrations of 20–40 micromolar, these in vitro findings may indeed hold clinical relevance, suggesting potential adverse effects at therapeutically achievable or elevated drug levels. Therefore, the potential clinical utility of protriptyline or its derivatives as a therapeutic agent specifically for human hepatoma warrants much more extensive and rigorous exploration in future research endeavors, taking into account both its efficacy and its cytotoxic profile.
In summary, this comprehensive investigation has elucidated several key aspects of protriptyline’s action in HepG2 human hepatoma cells. It has been established that protriptyline induces calcium influx through a distinct mechanism characterized as nifedipine-sensitive and non-store-operated, importantly, this process appears to be independent of protein kinase C activity. Simultaneously, protriptyline triggers the release of calcium from the endoplasmic reticulum, primarily through a phospholipase C-dependent pathway. Intriguingly, despite these significant alterations in calcium handling, the cell death induced by protriptyline in HepG2 cells is demonstrably dissociated from intracellular calcium rises, suggesting a calcium-independent cytotoxic mechanism. These detailed insights into protriptyline’s effects on calcium dynamics and its cytotoxicity in hepatoma cells are crucial considerations for any future in vitro or in vivo research seeking to understand or repurpose this compound.