Medicinal Chemistry, 2012, 8, 000-000
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Modulation of Signal-Transducing Function of Neuronal Membrane Na+,K+-ATPase by Endogenous Ouabain and Low-Power Infrared Radiation Leads to Pain Relief Ekaterina V. Lopatina1,2, Igor L. Yachnev1, Valentina A. Penniyaynen1, Vera B. Plakhova1, Svetlana A. Podzorova1, Tatiana N. Shelykh1, Ilya V. Rogachevsky1, Irina P. Butkevich1, Viktor A. Mikhailenko1, Anna V. Kipenko1,2 and Boris V. Krylov1,* Pavlov Institute of Physiology of Russian Academy of Sciences1, Nab. Makarova, 6, 199034, St. Petersburg, Russia Almazov Federal Heart, Blood and Endocrinology Centre2, St. Petersburg, Russia Abstract: Effects of infrared (IR) radiation generated by a low-power 2-laser on sensory neurons of chick embryos were investigated by organotypic culture method. Low-power IR radiation firstly results in marked neurite suppressing action, probably induced by activation of Na+,K+-Pase signal-transducing function. A further increase in energy of radiation leads to stimulation of neurite growth. We suggest that this effect is triggered by activation of Na+,K+-Pase pumping function. Involvement of Na+,K+-Pase in the control of the transduction process was proved by results obtained after application of ouabain at very low concentrations. Physiological significance of low-power IR radiation and effects of ouabain at nanomolar level was investigated in behavioral experiments (formalin test). It is shown that inflammatory pain induced by injection of formalin is relieved both due to ouabain action and after IR irradiation.
Keywords: Na+,K+-ATPase, signal-transducing function, patch-clamp, organotypic tissue culture, formalin test, ouabain, infrared radiation. METHODS
INTRODUCTION +
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The signal-transducing function of Na ,K -ATPase appears to have been acquired through the evolutionary incorporation of many specific binding motifs that interact with proteins and ligands. Binding of ouabain to Na+,K+-ATPase activates different signaling modules. On the one hand, the crosstalk between the activated pathways eventually modulates the expression of a number of genes [1]. That is why ouabain influences the growth of cardiac myocytes and the nerve tissue as well as proliferation of the smooth muscle cells; it also induces apoptosis in various malignant cells. On the other hand, it can control the excitability of slow sodium Nav1.8 channels that are responsible for pain sensation in mammals [2-4]. The coupling between Na+,K+-ATPase and Nav1.8 channels was shown earlier [5]. The hypothesis that the transducing function of Na+,K+-ATPase can play an important role in nociception requires more detailed investigation at membrane, cellular and behavioral levels. Supposedly, it is the signal-transducing function of Na+,K+-ATPase that is controlled by nanomolar concentrations of ouabain detected in human blood [6]. Application of these low concentrations of ouabain as well as low-power IR radiation is a delicate and efficient method to adequately verify our suggestions concerning the control of Na+,K +-ATPase signaltransducing function.
*Address correspondence to this author at the Pavlov Institute of Physiology of Russian Academy of Sciences, Nab. Makarova 6, 199034, St. Petersburg, Russia; Tel: +7 812 3281301; Fax: +7 812 3280501: E-mail:
[email protected] 1573-4064/12 $58.00+.00
Patch-clamp technique. Experiments were performed on short-term cultured isolated dorsal root ganglion (DRG) neurons from newborn Wistar rats. These nociceptive neurons are the small dark neurons with high density of Nav1.8 channels [7]. Dorsal ganglia were isolated from the L5-S1 region of the spinal cord and were placed in Hank’s solution. Enzymatic treatment [8] was performed for 8 min at 37 °C in a solution containing 1 ml Hank’s solution, 1 ml Eagle's medium, 2 mg/ml type 1A collagenase, and 1 mg/ml pronase E. The buffer used was 1 mM HEPES Na, pH 7.4. After this procedure, ganglia were thoroughly washed by centrifugation (1 min, 900 rpm) with changes of the supernatant solution. Washing and cultivation were performed using the solution consisting of Eagle's medium with glutamine based on Earle's solution (1:1), embryo calf serum (10%), glucose (0.6%), and gentamicin (40 U/ml). Mechanical dissociation was carried out by pipetting. The culture fluid was added to the resulting cell suspension to obtain the desired cell density in a plastic Petri dish. The non-neuronal cells were removed by allowing them to settle onto the surfaces of plastic 60-mm Petri dishes for 25 min at 37 °C, while the remaining cells, which were mostly the spinal ganglion neurons, were cultured on collagen-coated surfaces of 16-mm Petri dishes. Collagen was obtained from the rat tails. The extracellular solution contained 65 mM NaCl, 2 mM CaCl2, 2 mM MgCl2, 70 mM choline chloride, 10 mM HEPES Na, and 0.0001 mM tetrodotoxin (TTX), pH 7.4. The intracellular solution contained 100 mM CsF, 10 mM NaCl, 40 mM CsCl, 2 mM MgCl2, and 10 mM HEPES Na, © 2012 Bentham Science Publishers
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voltage clamping (Eerr) were taken into account by using the empirical formula Eerr INamax·Rs which determines the right-shift of all voltage-dependent functions. When the amplitude of the sodium current (INamax) was less than 1 nA, the series resistance error did not exceed 2 mV and was neglected.
Fig. (1). Photomicrograph of the dorsal root ganglia of 10-day old chicken embryos. Magnification, x100. A, control; B, after influence of IR radiation (qn = 2.210-10 Jcm-2).
pH 7.2. Exclusion of potassium ions allowed removing all the components of potassium currents; the fluoride ions were used in the intracellular solution to block the calcium currents [8]. All reagents were from Sigma. The volume of experimental bath was 200 μl. The external solution was changed four times every 3 min by passive flow under gravity. All experiments were conducted at room temperature of 22-24 °C. The integral ion currents were recorded in the whole-cell patch-clamp configuration [9]. Experiments were controlled by a hardware-software complex consisting of an EPC-7 amplifier, IBM-compatible personal computer, and an original software for automated control of the experiments. The series resistance (Rs) was constantly monitored in all the experiments and maintained below 2 . This parameter is extremely important in the quantitative studies of the changes in effective charge (Zeff) of Nav1.8 channels, because it not only determines the dynamic and stationary errors of the method [10], but also strongly affects the accuracy of Zeff measurements. In addition to degrading the accuracy of the method, the series resistance shifts the examined characteristics along the voltage axis. The stationary errors in
Organotypic tissue culture method. Chick embryos were received from Sinyavino Poultry (Leningrad District, Russia). The effects of ouabain and IR radiation on neurite growth of chick embryos sensory ganglia (10–12 days) were studied in organotypic tissue culture. Studies were performed on 700 explants. The ganglia were cultured for three days on collagen supports in Petri dishes at 36.5 °C and 5% CO2. CO2 incubator (Sanyo, Japan) was used. The nutrient medium contained 40% Hank’s solution, 40% Eagle’s medium, 5% chick embryo extract, and 15% fetal calf serum, and was supplemented with insulin (0.5 U/ml), glucose (0.6%), glutamine (2 mM), and gentamicin (100 U/ml). Explants not exposed to action of IR radiation or ouabain served as the control. The time of exposition of IR radiation was 3 min. Ouabain (selective inhibitor of Na+,K +-ATPase; Sigma) was added to the culture medium. The growth of explants in tissue culture was controlled on vital preparations using a confocal laser scanning microscope LSM-710 (Carl Zeiss, Germany). The preparations were also visualized by a television microscope attachment (Carl Zeiss Axiostar Plus, Germany). Neurite outgrowth was quantified using the PhotoM 1.2 image analysis software designed by A. Chernigovskiy (Ioffe Physical-Technical Institute, Russian Academy of Sciences, St. Petersburg, Russia). The area index (AI) was used to estimate the neurite outgrowth. It was calculated in relative units as follows: AI = GZ/CZ, where CZ is the central zone area (the initial area of the ganglion body) and GZ is the peripheral growth zone (Fig. 1). AI value was taken as 100% for the control experiments. Behavioral tests. Experiments were performed on adult male Wistar rats, weighing 200 – 220 g, bred in the vivarium of Pavlov Institute of Physiology, St. Petersburg, Russia. Animals were kept under standard controlled laboratory conditions (temperature 22 ± 2 °C, relative humidity 50% and 12/12 hours light/dark cycle with the light switched on at 9 a.m.), and housed individually with free access to standard pelleted food and tap water. Physiological significance of low-power IR radiation generated by a CO2-laser and of effects of ouabain at nanomolar level was investigated in behavioral experiments in the formalin test in 72-78-day-old male rats (n = 19 and n = 14, respectively). A dilute formalin solution (2.5%, 50 l) was injected into the plantar pad of the left hind paw. The response to injection of formalin is presented by pain-related patterns, licking (the supraspinal level) and flexing/shaking (the spinal level) of injected paw, which were registered during 90 min using a self-made computer program. The formalin test, an inflammatory pain model, provides a way of evaluating two types (acute and tonic) of pain that are seen in the first and second phases, respectively, of the response to the externally applied stimulus [11, 12]. The first phase lasts for about 5 min and is considered to be a direct effect of formalin on the peripheral nociceptive endings. The second phase (about 50-90 min) is considered to result from the changes in the central nervous system function induced by neural activity generated during
Modulation of Signal-Transducing Function
the first phase and from the developing process of inflammation caused by the stimulus during the second phase. Between the phases there is the interphase, the period without any pain-related behaviors. Intraperitoneal injections of ouabain (0.3 mg/kg, 1 ml) have been performed 10 min prior to the formalin test. Experiments with IR radiation were carried out in conditions of faint lighting. After injection of formalin, rats were treated with radiation of power density 9.010-4 Wcm-2 during 7 min. Motor reactions of each rat were restricted with a
Medicinal Chemistry, 2012, Vol. 8, No. 1
where G Namax and GNa(E) are the maximal value and voltage dependence of the chord conductance, respectively. G Na(E) could be obtained in the patch-clamp experiments as GNa(E) = Imax(E)/(E – ENa), where Imax is the amplitude of the sodium current, ENa is the reversal potential for sodium ions. GNa(E) is a monotonous function which approaches its maximum value GNamax at positive potentials E. According to the Almers theory, the limiting-slope procedure can be applied:
lim(No/Nc) = lim{G Na(E)/[G Namax – GNa(E)]} C·exp[(Zeffe0 E)/(kT)], E - E - E - bell glass. The intensity of biphasic response to formalin injection (the number of flexes+shakes and the time spent licking in each phase) and the duration of both phases and the interphase (min) were analyzed. Each rat was tested once only. The Ethical Guidelines issued by the Committee of the International Association for the Study of Pain have been followed [13]. All experimental procedures were in full compliance with The European Council Directive (86/609/EEC) and approved by The Ethical Committee of the Pavlov Institute of Physiology.
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(Equation 1)
where No is the number of open channels, Nc is the number of closed channels at maintained potential E. The slope of the asymptote passing through the first points determined by the most negative values of the membrane potential makes it possible to estimate the Zeff value (Equation 1). It can be done because at these potentials the Boltzmann’s principle is applicable, where k is the Boltzmann constant, T is the absolute temperature, C is a constant, e0 is the electron charge.
Calculational methods. The full conformational search of ouabain molecule and molecules of ouabain–Ca2+ chelate complexes was performed by RHF method with 6-31G* basis set [14] within GAMESS program package [15]. Firstly, all possible ouabain conformations (4) were obtained. The sets of geometrical and electronic parameters of all chelate complexes conformations were calculated as follows. The conformations of free ouabain molecule served as the starting points. All pairwise combinations of oxygen atoms of ouabain capable of chelating Ca2+ (6 atoms, 15 pairs) were picked one after the other, Ca2+ ions were positioned exactly in the middle of the line connecting the oxygen atoms in a pair, and full geometry optimizations of ouabain–Ca2+ systems were further performed. Thus, 60 optimization were carried out (15 pairs, 4 ouabain conformations). Statistical analysis. The data were processed with Student’s t test at 0.05. RESULTS AND DISCUSSION Investigation of Na+,K+-ATPase Signal-transducing Function at the Membrane Level. Role of Endogenous Ouabain. Patch-clamp data. Rat DRG nociceptive neuron membrane was investigated using the whole-cell patch-clamp method. Extracellular application of ouabain leads to the decrease of the effective charge transfer (Zeff) in the activation gating system of Nav1.8 channels. The limiting-slope procedure [16] is used in our investigation to estimate Zeff. The approach has been described in our publications [5, 17]. The ratio of the number of open channels (No) to the number of closed channels (Nc) is calculated as No/Nc = G Na(E)/[G Namax – GNa(E)],
Fig. (2). Relationship between the effective charge Zeff (electron charge units) and extracellular ouabain concentration (M). Means and SEM represent results obtained in several experiments, the numbers of which (n) are shown below each bar. *Significant differences compared with control experiments, p
