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11BIOLOGICAL SCIENCES
EFFECT OF CALCIUM IONS AND CHLORPROMAZINE ON TRPV1 CHANNELS OF RAT DRG
NEURONS
Petrushenko M.,
Junior scientific researcher Petrushenko O.,
Senior scientific researcher, Lukyanetz E.
Professor, Dr.Sci Department of Biophysics of Ion Channels, O.O. Bogomolets Institute of Physiology, NAS of Ukraine,
Kyiv, Ukraine
Abstract
We investigated the effect of the calmodulin blocker chlorpromazine on the activation of TRPV1 channels caused by the application of capsaicin in solutions with low and high concentrations of external calcium. The experiments were performed on a 1-2-day primary culture of neurons isolated from the dorsal root ganglia (DRG) of rats aged 9-12 days. To determine the level of intracellular calcium in the experiments, we used the method of microfluorimetry. Neurons were loaded with a fluorescent probe Fura 2AM. Small neurons belonging to nocicep-tors with a diameter of 15-37 ^m, the surface area averaged 590 ^m2, were selected for the experiment. Capsaicin (0.5 ^M) was applied for 20 s. Chlorpromazine was administered for 2 min, the second application of capsaicin was performed 1 min after the chlorpromazine administration. All observed responses to capsaicin could be divided according to the response rate - with the rapid and slow development of the effect of capsaicin. We have shown that the action of the calmodulin blocker chlorpromazine depends on the concentration of Ca2+ ions in the external solution. The latter depends on the increase in the concentration of Ca2+ in the solution. At low concentrations of external Ca2+ (0.25 mM), calmodulin enhances the effect of capsaicin on TRPV1 channels in cells with a slow development of the effect of capsaicin, and at high concentrations of Ca2+ (2.5 mM) causes non-competitive inhibition of Ca2+ entry through TRPV1 channels. We hypothesize that the intracellular calcium level determines the effect of chlorpromazine on TRPV1 channels.
Keywords: calcium, TRPV1 channels, capsaicin, chlorpromazine.
Introduction
It is known that calcium intracellular signaling is widely involved in regulating numerous processes and reactions, including in the regulation of calcium itself into the cell through a variety of ion channels, including some TRP channels (Kostyk and others 2005; Kostyk and others 2010; Kostyuk and Lukyanetz 2006). It was shown that entry of Ca2+ into the cell through TRPV1 channels under the influence of capsaicin (Caps), agonist of TRPV1 channels, causes their desensitization (Caterina and others 1997; Koplas and others 1997; Petrushenko and others 2020; Petrushenko and others 2021; Petrushenko and Luk'yanetz 2019; Yang and others 2014). On the other hand, the Ca2+ application to the intracellular surface of the membrane leads to a decrease in Caps-induced current due to calmodulin-me-diated events (Rosenbaum and others 2004).
Calmodulin (CM) binds Ca2+ and serves as a regulator of Ca2+-dependent processes in the cell. Ca2+-bound calmodulin usually inhibits several Ca2+ channels with a dangerous increase in intracellular Ca2+ through calcineurin activation and protects cells from Ca2+- cytotoxicity, so blocking calmodulin should lead to an uncontrolled increase in intracellular Ca2+ (Caterina and others 1997; Doroshenko and others 1988). This uncontrolled increase in Ca2+ should lead to inhibition of TRPV1 channels.
CaM is actively involved in modulating the activity of various components of the cell through direct action or by activating several Ca2+/Ca2+-dependent enzymes that regulate many ion channels, including potential-dependent calcium ion channels (Doroshenko and others 1988).
Materials and methods
The method of microfluorescent microscopy was used to determine the level of intracellular calcium in the experiments. The experiments were performed on a 1-2 day primary culture of neurons isolated from the DRG of rats. Neurons were stained for 30 min with a fluorescent probe Fura 2 AM (1 ^M).
1. Isolation of DRG neurons and obtaining the primary culture.
Wistar rats of both sexes were used in the experiments. Animals aged 9-12 days and weighing 15-25 grams were taken into the experiment. Animal experiments were conducted following existing international and national regulations on the use of experimental animals, in particular: the Council of Europe Convention of 18.03.1986 and the Law of Ukraine of 21.02.2006 №» 3447-IV.
In our experiments, the type of neurons was determined visually by estimating the diameter of isolated cells in the image, Fig.1.
w
" i N i •
Fig. 1A photomicrograph showing the size of the studied DRG neurons is presented. There are two neurons in
the middle.
In this case, to minimize the possible error, cells with a diameter of not more than 12-35 ^m were selected. Isolation of DRG neurons and obtaining the primary culture were performed as described (Dragan and others 2016; Petrushenko 2013). In short: the isolated ganglia were transferred to solution 1 for enzymatic treatment. Treated with a mixture of enzymes tryp-sin/collagenase in a ratio of 3: 1 (3 mg/ml trypsin, 1 mg/ml collagenase) for 35-38 minutes at 34 ° C. After enzymatic treatment, the ganglia were transferred to the culture solution and washed twice with the enzyme. The ganglia were then transferred to a drop of the medium on cover slides in Petri dishes (approximately 6 slides per 1 cup with a volume of 3 ml) and mechanically dispersed with glass micropipettes and metal needles. The neurons released due to this procedure remained on the slides for 30-40 minutes in a CO2 incubator. Next, 2.5 ml of culture medium was added to each cup. As such, the obtained primary culture of DRG neurons was in a CO2 incubator and cells were used in experiments for 20-72 h after preparation.
2. Experimental solutions and media for cell culture.
Solution for isolation - aqueous solution on phosphate buffer DPBS (D5773) with the addition (mM): CaCl2-1, HEPES-20, NaHCOs-15. The pH of the solution was adjusted to 7.32 with NaOH.
The culture medium contained 90% liquid DMEM medium and 10% inactivated calf embryo serum. In the resulting solution, 26 mm NaHCO3, 0.6 ^m dry bovine insulin and a mixture of Peniciline/Streptomicine (0.03%) were added. All reagents used to prepare the above solutions were purchased from Sigma-Aldrich, USA.
Base solution for experiments - aqueous solution containing (in mM): NaCl - 150; KCl - 5; MgCl2 - 1.4, glucose - 5, HEPES - 20. The pH of the solution was adjusted to 7.35-7.40 with NaOH. The desired concentration of CaCl2 (1 - 3 mM) was added under experimental conditions.
3. Measurement of intracellular level of Ca2+ (Calcium imaging)
The intracellular calcium level was measured using a fluorescent indicator Ca2+ - Fura 2AM, as described earlier (Dragan and others 2016). The reduction of Ca2+ was carried out using a selective chelator of Ca2+ ions - BARTA. Neurons were loaded with Fura-2AM in a solution containing Ca2+ and Mg2+ ions to stabilize the cell membrane and ensure dye fluorescence. The solution in the experimental chamber did not contain divalent cations. At the same time, the solution for applying depolarizing concentration of KCl and capsaicin contained 1 mM Ca2+ and 1 mM Mg2+.
Results and discussion
Caps-positive neurons belong to the subpopulation of DRG neurons with a small soma diameter (<50 ^m) with thin unmyelinated fibers (C). According to (Caterina and others 1997), Caps-activated cation channels are localized in small DRG neurons. However, in some tissues, such as tooth pulp, large-diameter neurons with A-fibers may also respond to Caps (Ikeda and others 1997). In our studies, the vast majority of Caps-sensitive neurons belonged to a subpopulation of small-diameter neurons.
During long-term applications of Caps on the neuron, TRPV1 receptors responded by reducing the activity - the amplitude of calcium transients was decreased. This phenomenon is called desensitization. Desensiti-zation of TRPV1 channels by type of tachyphylaxis reduces the maximum calcium transient amplitude during successive additions of the same concentrations of cap-saicin (Koplas and others 1997). Desensitization to the influence of Caps in DRG neurons lasts 20-45 minutes after achieving tachyphylaxis (Mandadi and others 2004).
Rosenbaum and coworkers have shown that Caps activated an input current with an ED50 of 728 nM, demonstrating co-operation (Hill's coefficient, 2.2); however, both forms of desensitization were weakly dependent on [Caps], suggesting a separation between the activation of Caps-sensitive channels and desensiti-zation (Rosenbaum and others 2004).
[Ca2+J
Fig.2 Dependence of the amplitude of Caps-induced calcium transients on the concentration of Ca2+ in the external solution in the presence of 70 ]uM chlorpromazine and without it. The mean values of the amplitude is
presented in relative units.
We have shown that the effect of calmodulin blocker chlorpromazine (50-100 ^M) increases intracellular Ca2+ concentration and depends on the concentration of Ca2+ ions in the external solution. At low concentrations of external Ca2+ (0.25 mM) in cells with a slow development of capsaicin effect, calmodulin enhances the effect of capsaicin on TRPV1 channels, and at high concentrations of Ca2+ (2-3 mM) causes noncompetitive inhibition of TRPV1 channels and reduced Ca2+ entry. The entry of Ca2+ through TRPV1 channels follows the kinetics of Michaelis-Menten. Calculated Km = 750 ^M (Fig. 2).
TRPV1 channels are characterized by bicompo-nent desensitization: fast, which occurs immediately after the entry of calcium into the cytosol, and a calcium-independent slow component, characterized by the involvement of endogenous regulatory molecules. As a result of repeated applications of the same concentration of capsaicin, desensitization of the TRPV1 channel may develop, which manifests itself in a partial decrease in the maximum amplitude of the response (tachyphylaxis) or complete inactivation, Fig.3.
desensitization
4
inactivation
Fig.3. Inhibition of TRPV1 channel functions.
Studies (Rosenbaum and others 2004) have shown that in Ca2+/CaM decreased Caps activated current. The use of Ca2+ with CaM enhanced the inhibitory effect of Ca2+ and led to a much more significant reduction in current. In works of Numazaki and coauthors (Numazaki and others 2003), it was shown that Ca2+-
binding protein calmodulin binds to the 35-a-segment at the C-terminus of the TRPV1 channel, and disruption of this calmodulin-binding segment prevents desensitization of TRPV1.
One of the calcium-dependent enzymes in the cell that can regulate ion channels is calcineurin. This enzyme is involved in calcium-dependent inactivation of potential-dependent membrane channels (Kostyuk and Lukyanetz 1993; Lukyanetz and others 1998). Previous studies of TRPV1 desensitization have shown that cal-cineurin inhibitors reduce this desensitization, indicating the involvement of the Ca2+-dependent phosphory-lation/ dephosphorylation process.
It has been shown that the desensitization of TRPV1-WT currents activated by capsaicin is significantly reduced by the complex of cyclosporin A with cyclophilin, which is a specific inhibitor of protein phosphatase 2B (calcineurin) (Docherty and others 1996). The following mechanism of capsaicin desensi-tization sensitivity has also been proposed: growth of [Ca2+]i and activates calcineurin, which leads to dephosphorylation and desensitization of capsaicin-sensitive ion channels (Olah and others 2007).
It can be suggested that in our experiments, the inhibitory effect of chlorpromazine on calmodulin-de-pendent phosphatase also depended on the influx of Ca2+. In solutions with high Ca2+ levels, there is a decrease in the blocking effect of chlorpromazine on protein phosphatase and increased inactivation of the TRPV1 channels. In solutions with low Ca2+ levels, the blocking effect of chlorpromazine on calcineurin is increased, which leads to a decrease of the TRPV1 channel inactivation and increased input of Ca2+ through the channels. Interestingly, the double action of the intracellular action of calcium - on the one hand, activating, and on the other - depressing, depending on the concentration of these ions, was observed in the case of potentially dependent calcium channels (Shkryl and others 2001).
Ca2+-bound calmodulin usually inhibits several types of Ca2+ channels with a dangerous increase in intracellular Ca2+ and protects cells from Ca2+ -cytotoxi-city, so blocking calmodulin should theoretically lead to an uncontrolled increase in intracellular Ca2+ (Doroshenko and others 1988; Olah and others 2007).
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