Caffeine Suppresses GABA Receptor-Mediated Current in Rat Primary Sensory Neurons Via Inhibition of Intracellular Phosphodiesterase

In acutely isolated rat primary sensory neurons, the effects of caffeine on GABA receptormediated current (IGABA) were investigated using a whole-cell patch clamp technique. We found that applications of GABA (10-1000 µM) induced inward currents in a concentration-dependent manner; the currents ma...

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Datum:	2015
Hauptverfasser:	Yang, J.Y., Yang, G., Ren, J., Zhao, J., Li Sh.
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Sprache:	English
Veröffentlicht:	Інститут фізіології ім. О.О. Богомольця НАН України 2015
Schriftenreihe:	Нейрофизиология
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spelling	irk-123456789-1481862019-02-18T01:23:14Z Caffeine Suppresses GABA Receptor-Mediated Current in Rat Primary Sensory Neurons Via Inhibition of Intracellular Phosphodiesterase Yang, J.Y. Yang, G. Ren, J. Zhao, J. Li Sh. In acutely isolated rat primary sensory neurons, the effects of caffeine on GABA receptormediated current (IGABA) were investigated using a whole-cell patch clamp technique. We found that applications of GABA (10-1000 µM) induced inward currents in a concentration-dependent manner; the currents manifested obvious desensitization. Pretreatment with caffeine (0.01- 100 µM) suppressed IGABA in a noncompetitive manner; caffeine shifted the concentration – response curve for GABA downwards compared to the control. Theophylline showed a similar and stronger inhibitory effect on IGABA. Isolated application of 1 µM diazepam enhanced IGABA, while pretreatment with 10 µM caffeine and 1 µM diazepam suppressed this current. Intracellular application of the protein kinase A inhibitor H-8 dramatically weakened the inhibitory effect of caffeine on IGABA. Because primary afferent depolarization is related to GABAA receptors, our results suggest that caffeine might antagonize presynaptic inhibitory effects of primary afferents, probably via inhibition of intracellular phosphodiesterase. Досліджували впливи кофеїну на струми, опосередковані рецепторами ГАМК (IGABA), в гостроізольованих первинних нейронах щура; використовували методику петчклемп у конфігурації «ціла клітина». Аплікації ГАМК (10–1000 мкМ) викликали струми вхідного напрямку, амплітуда котрих була залежною від концентрації; струми демонстрували яскраво виражену десенситизацію. Попередня обробка кофеїном (0.01–100 мкМ) призводила до неконкурентного пригнічення IGABA. Вплив кофеїну зміщував вниз криву концентрація–відповідь для ГАМК порівняно з контролем. Теофілін демонстрував подібні й навіть сильніші гальмівні впливи на IGABA. Ізольована аплікація 1 мкМ діазепаму посилювала IGABA, тоді як попередня обробка 10 мкМ кофеїну перед дією 1 мкМ діазепаму пригнічувала цей струм. Внутрішньоклітинна аплікація H-8 (інгібітору протеїнкінази А) дуже значно послаблювала гальмівний вплив кофеїну на IGABA. Оскільки деполяризація терміналий первинних аферентів опосередковується рецепторами ГАМК, наші результати вказують на те, що кофеїн може протистояти пресинаптичному гальмуванню первинних аферентів. Ефекти кофеїну опосередковуються гальмуванням внутрішньоклітинної фосфодіестерази. 2015 Article Caffeine Suppresses GABA Receptor-Mediated Current in Rat Primary Sensory Neurons Via Inhibition of Intracellular Phosphodiesterase / J.Y. Yang, G. Yang, J. Ren, J. Zhao, Sh. Li // Нейрофизиология. — 2015. — Т. 47, № 2. — С. 129-135. — Бібліогр.: 25 назв. — англ. 0028-2561 http://dspace.nbuv.gov.ua/handle/123456789/148186 612.822:612.015.1 en Нейрофизиология Інститут фізіології ім. О.О. Богомольця НАН України
institution	Digital Library of Periodicals of National Academy of Sciences of Ukraine
collection	DSpace DC
language	English
description	In acutely isolated rat primary sensory neurons, the effects of caffeine on GABA receptormediated current (IGABA) were investigated using a whole-cell patch clamp technique. We found that applications of GABA (10-1000 µM) induced inward currents in a concentration-dependent manner; the currents manifested obvious desensitization. Pretreatment with caffeine (0.01- 100 µM) suppressed IGABA in a noncompetitive manner; caffeine shifted the concentration – response curve for GABA downwards compared to the control. Theophylline showed a similar and stronger inhibitory effect on IGABA. Isolated application of 1 µM diazepam enhanced IGABA, while pretreatment with 10 µM caffeine and 1 µM diazepam suppressed this current. Intracellular application of the protein kinase A inhibitor H-8 dramatically weakened the inhibitory effect of caffeine on IGABA. Because primary afferent depolarization is related to GABAA receptors, our results suggest that caffeine might antagonize presynaptic inhibitory effects of primary afferents, probably via inhibition of intracellular phosphodiesterase.
format	Article
author	Yang, J.Y. Yang, G. Ren, J. Zhao, J. Li Sh.
spellingShingle	Yang, J.Y. Yang, G. Ren, J. Zhao, J. Li Sh. Caffeine Suppresses GABA Receptor-Mediated Current in Rat Primary Sensory Neurons Via Inhibition of Intracellular Phosphodiesterase Нейрофизиология
author_facet	Yang, J.Y. Yang, G. Ren, J. Zhao, J. Li Sh.
author_sort	Yang, J.Y.
title	Caffeine Suppresses GABA Receptor-Mediated Current in Rat Primary Sensory Neurons Via Inhibition of Intracellular Phosphodiesterase
title_short	Caffeine Suppresses GABA Receptor-Mediated Current in Rat Primary Sensory Neurons Via Inhibition of Intracellular Phosphodiesterase
title_full	Caffeine Suppresses GABA Receptor-Mediated Current in Rat Primary Sensory Neurons Via Inhibition of Intracellular Phosphodiesterase
title_fullStr	Caffeine Suppresses GABA Receptor-Mediated Current in Rat Primary Sensory Neurons Via Inhibition of Intracellular Phosphodiesterase
title_full_unstemmed	Caffeine Suppresses GABA Receptor-Mediated Current in Rat Primary Sensory Neurons Via Inhibition of Intracellular Phosphodiesterase
title_sort	caffeine suppresses gaba receptor-mediated current in rat primary sensory neurons via inhibition of intracellular phosphodiesterase
publisher	Інститут фізіології ім. О.О. Богомольця НАН України
publishDate	2015
url	http://dspace.nbuv.gov.ua/handle/123456789/148186
citation_txt	Caffeine Suppresses GABA Receptor-Mediated Current in Rat Primary Sensory Neurons Via Inhibition of Intracellular Phosphodiesterase / J.Y. Yang, G. Yang, J. Ren, J. Zhao, Sh. Li // Нейрофизиология. — 2015. — Т. 47, № 2. — С. 129-135. — Бібліогр.: 25 назв. — англ.
series	Нейрофизиология
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first_indexed	2025-07-12T18:33:12Z
last_indexed	2025-07-12T18:33:12Z
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fulltext	NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2015.—T. 47, № 2 129 UDC 612.822:612.015.1 J. Y. YANG1, G. YANG2,3, J. REN4, J. ZHAO2, and Sh. LI2 CAFFEINE SUPPRESSES GABA RECEPTOR-MEDIATED CURRENT IN RAT PRIMARY SENSORY NEURONS VIA INHIBITION OF INTRACELLULAR PHOSPHODIESTERASE Received December 30, 2013 In acutely isolated rat primary sensory neurons, the effects of caffeine on GABA receptor- mediated current (IGABA) were investigated using a whole-cell patch clamp technique. We found that applications of GABA (10-1000 µM) induced inward currents in a concentration-dependent manner; the currents manifested obvious desensitization. Pretreatment with caffeine (0.01- 100 µM) suppressed IGABA in a noncompetitive manner; caffeine shifted the concentration – response curve for GABA downwards compared to the control. Theophylline showed a similar and stronger inhibitory effect on IGABA. Isolated application of 1 µM diazepam enhanced IGABA, while pretreatment with 10 µM caffeine and 1 µM diazepam suppressed this current. Intracellular application of the protein kinase A inhibitor H-8 dramatically weakened the inhibitory effect of caffeine on IGABA. Because primary afferent depolarization is related to GABAA receptors, our results suggest that caffeine might antagonize presynaptic inhibitory effects of primary afferents, probably via inhibition of intracellular phosphodiesterase. Keywords: GABA receptor-mesiated current, primary sensory (DRG) neurons, whole- cell patch clamp, caffeine, phosphodiesterase. 1 Department of Urology Dalian Friendship Hospital, Dalian, China. 2 Department of Physiology. 3 Department of Clinical Medicine, Anesthesiology Major, Dalian Medical University, Dalian, China. 4 Department of Anesthesiology, Jinan Central Hospital, Jinan, China. Correspondence should be addressed to Sh. Li (e-mail: lishao89@hotmail.com), J. Zhao (e-mail: zhaoj@dlmedu.edu.cn), J. Y. Yang (e-mail : yangjinyi90@hotmail.com). INTRODUCTION Caffeine and theophylline are methylxanthine alkalo- ids. These drugs exert a number of pharmacological effects; they antagonize asthma, relax smooth muscles, excite the CNS and cardiac muscle, influence diuresis, etc. [1-3]. The mechanisms underlying these effects, according to the data available, may include antagonism with respect to the adenosine receptors [4-6], inhibition of phosphodiesterase [7, 8], acceleration of the release of intracellular calcium [9, 10], competitive action on benzodiazepine receptors, and influence on GABA receptor-mediated effects [3, 11, 12]. GABA is one of the primary inhibitory neurotransmitters in the spinal cord and brain; it induces depolarization of terminals of primary afferent fibers and, due to this, reduces the release of an excitatory transmitter from primary afferent neurons providing, in such a way presynap- tic inhibition. Our study was aimed at an investigation of the effects of caffeine on GABAA receptor-mediated responses in acutely isolated rat primary sensory neurons (dorsal root ganglion neurons, DRG neurons). We believe that our findings may throw light on the understanding of the mechanism underlying modulation of the activity of primary afferents. METHODS Isolation of DRG Neurons. Two- to three-week- old Sprague–Dawley rats of both sexes (n = 18) were obtained from the Faculty of Laboratory Animal Science, Dalian Medical University. The rats were anaesthetized with ether and then decapitated. The thoracic and lumbar segments of the vertebral column were dissected and longitudinally divided into two halves along the median line on both dorsal and ventral sides. Dorsal root ganglions (DRGs), together with the dorsal and ventral roots and attached spinal nerves, were taken out and transferred immediately into Dulbecco’s modified Eagle’s medium (DMEM) NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2015.—T. 47, № 2130 J. Y. YANG, G. YANG, J. REN et al. at pH 7.4. After the removal of attached nerves and surrounding connective tissues, the DRGs were minced with fine spring scissors, and ganglion fragments were placed in a flask containing 5 ml DMEM with trypsin (type III), collagenase (type I), and collagenase (type II; 0.5 mg/ml each) added, and incubated at 35°C for 30-35 min in a shaking water bath. Soybean trypsin inhibitor (type II-s, 1.25 mg/ml) was then added to stop trypsin digestion. Dissociated neurons were placed into a 35-mm Petri dish and kept for at least 30 min before electrophysiological recording. The somata of neurons selected for recording were 15-45 μm in diameter. All reagents mentioned above were from Sigma (USA) Electrophysiological Recordings. Whole-cell patch clamp recording was carried out using an EPC- 10 amplifier (HEKA Electronik, Germany). Pulse and PulseFit 8.6 software programs were used for data acquisition and analysis. Electrodes were pulled from borosilicate glass capillaries on a micropipette puller (Narrishige, PP 830, Japan); their resistance was 3-6 MΩ when filled with an internal solution contain- ing (mM): KCl, 140; MgCl2, 2.5; Hepes, 10; EGTA, 11; ATP, 5 (osmolarity adjusted to 320 mosmol/l with sucrose; pH adjusted to 7.2 with KOH). Cells were bathed in an external solution containing (mM): NaCl, 150; KCl, 5; CaCl2, 2.5; MgCl2, 2; Hepes, 10; D-glu- cose, 10 (osmolarity adjusted to 320 mosmol/l with sucrose; pH adjusted to 7.4 with NaOH). A small patch of the membrane underneath the tip of the pipette was aspirated to form a gigaohm seal (1-10 GΩ), and then a more subatmospheric pressure was applied to rupture it, thus establishing a whole-cell configura- tion. Currents were filtered at 1 kHz and digitized at 104 sec–1. Adjustment of capacitance compensation and series resistance compensation was done before recording the membrane currents. The leak current was digitally subtracted. Experiments were carried out with the membrane potential held at –60 mV. All recordings were performed at room temperature (22-24°C). Intracellular Dialysis using the Repatch Technique. For the repatch experiment, the first patch was used as the control, with the pipette filled with the normal internal solution. After the current had been recorded, the pipette was discarded. On the same neuron, the second patch was performed using another pipette that was filled with the internal solution containing H-8 (N-[2-(methylamino)ethyl]- 5-isoquinoline sulfonamide ∙ 2HCl), an inhibitor of protein kinase A. After 30 min, the membrane current was recorded again and compared with the control one. Drug Application. Drugs used in the experiments, caffeine, GABA, theophylline, diazepam, and H-8 (all from Sigma, USA) were dissolved in external or internal solutions just before use. All drugs except for H-8 were dissolved in the external solution and applied by gravity flow from a linear array of fused silica tubules (external and internal diameters 500 and 200 μm, respectively) connected to a series of independent reservoirs. The distance from the tubule mouth to the cell examined was about 100 μm. Application of each drug was driven by gravity and controlled by the corresponding valve, and rapid solution exchange could be achieved within about 100 msec by shifting the tubes horizontally with a micromanipulator. Cells were constantly bathed between drug applications in the normal external solution flowing from one tube connected to a larger reservoir. In the experiments where H-8 was applied intracellularly, it was dissolved in the internal solution before use. The interval between GABA applications was at least 4 min, in order to avoid desensitization. Statistics. Numerical data were statistically compared using the Student’s t-test or ANOVA. Statistical analysis of the concentration–response data was performed using a nonlinear curve-fitting program, ALLFIT. The values for currents are expressed as means ± s.e.m. Differences with P < 0.05 were considered significant. RESULTS Freshly isolated neurons from the rat DRGs had a round or oval shape with a residue of the stem process, which was cut off by enzymatic and mechanical treatment. Experiments were carried out on 146 freshly isolated DRG neurons, the diameters of which varied from 15 to 45 μm. GABA Receptor-Mediated Current. In the majority of the neurons examined (138/146, 94.5%), GABA (10 – 1000 µM) induced inward currents in a dose-dependent manner (Fig. 1A). This GABA receptor-mediated current (IGABA, 300 µM) could be mimicked by 300 μΜ muscimol (B) and blocked by 300 µM bicuculline, a selective antagonist of GABAA receptors, which indicated that this current was mediated by the GABAA receptors (C). This kind of current exhibited an apparent desensitization, especially at high concentrations. When GABA was applied repetitively for 20 sec with 4-min-long intervals, IGABA was stable for at least 90 min, and the NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2015.—T. 47, № 2 131 CAFFEINE SUPPRESSES GABA RECEPTOR-MEDIATED CURRENT caffeine applications was used in the subsequent study. Concentration–Response Relationship for Caffeine Inhibition of GABA Receptor-Mediated Current. Figure 3 shows the concentration–response curves for IGABA in the absence (1) and presence (2) of 100 µM caffeine (n = 9). All responses were normalized with respect to the peak current induced by 1000 µM GABA alone. Caffeine (10 µM) shifted the concentration–response curve of IGABA downward markedly, and the maximal IGABA decreased to 57% of its control. The EC50 values for both curves were very close (28 vs 30 µM) and showed no statistical difference. Inhibitory Effect of Theophylline on GABA Receptor-Mediated Current. The modulatory effects of 10 µM caffeine and 10 µM theophylline on GABA receptor-mediated current in the same DRG neurons were compared. Both these drugs induced inhibition of IGABA, and the mean inhibition rates were 24.5 ± ± 8.7% (n = 9) and 41.1 ± 11.0% (n = 9), respectively (data for each group are from the same DRG neurons). Thus, theophylline exerted a noticeably stronger A A B 10 μM 0.01 0.01 0.1 0.1 1 1 10 20 30 40 60 50 0 10 100 μM 10 GABA GABA GABA control control washout muscimol 30 μM 100 μM 300 μM 300 μM 300 μM 400 pA 400 pA 30 sec 30 sec 300 μM bicuculline 100 μM caffeine 100 μM GABA 300 μM 1000 μM B C F i g. 1. GABA receptor-mediated currents (IGABA) in DRG neurons and their inhibition by bicuculline. A) Traces of IGABA induced by applications of GABA in different concentrations (10 – 1000 µM). B) Inward currents evoked by separate applications of 300 µM GABA and 300 µM muscimol. C) Inward current evoked by 300 µM GABA, and blocking of this current by the GABAA receptor antagonist bicuculline (300 µM). Р и с. 1. Струми, опосередковані рецепторами ГАМК, у нейронах дорсального ганглію та гальмування цих струмів бікукуліном. variation of its amplitude did not exceed 7.0% (data not shown). Thus, we used this pattern of GABA applications in the following experiments. Modulatory Effects of Caffeine on GABA- Activated Current in Rat DRG Neurons. When caffeine (0.01-100 µM) was applied for 1 min prior to application of GABA (100 µM), attenuation of IGABA was observed. The inhibitory effect of caffeine depended on the concentration of the latter. The current activated by 100 μΜ GABA was suppressed by 13.2 ± 4.8% (n = 9), 20.2 ± 3.1% (n = 9), 38.9 ± ± 6.1% (n = 9), 43.6 ± 5.5% (n = 9), and 44.3 ± 6.1% (n = 9) when 0.01, 0.1, 1, 10, and 100 µM caffeine were pre-applied, respectively. The concentration– response curve of caffeine inhibition of IGABA was S-shaped, and the IC50 value was 10 µM (Fig. 2). IGABA recovered quickly after caffeine-induced inhibition; complete recovery was observed in about 4 min (not shown). Therefore, a 4-min-long interval between F i g. 2. Inhibition of GABA receptor-mediated current (IGABA, 100 µM GABA) by 1-min-long pretreatment with caffeine. A) Suppression of IGABA induced by caffeine in different concentrations (0.01 – 100 µM) in the same DRG neuron. B) Dose-dependence curve of inhibition of IGABA (100 µM) by caffeine (0.01 – 100 µM). Р и с. 2. Пригнічення струму, опосередкованого рецепторами ГАМК – IGABA (100 мкМ ГАМК), після однохвилинної попередньої обробки кофеїном. NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2015.—T. 47, № 2132 J. Y. YANG, G. YANG, J. REN et al. inhibitory effect on IGABA than caffeine at the same concentration (P < 0.01, Fig. 4). Modulation by Diazepam of GABA Receptor- Mediated Current. In the same DRG neurons, IGABA (100 µM) was enhanced by 19.3 ± 2.2% with pretreatment by diazepam (10 µM, 60 sec; n = 9), whereas simultaneous application of diazepam and caffeine decreased IGABA by 26.0 ± 7.5% (n = 9) (Fig. 5). Dependence of Caffeine-Induced Inhibition of IGABA on the Pretreatment Time. As is shown in Fig. 6, caffeine did not suppress GABA receptor- mediated currents when this agent was applied to the DRG neurons simultaneously with GABA (i.e., with no pretreatment). However, when pre-application of caffeine lasted at least 20 sec, the inhibition of IGABA by this drug emerged clearly. This suggests that inhibition of IGABA by caffeine is a time-dependent process, and, thus, some intracellular signal transduction pathway may be involved. Caffeine-induced inhibition on IGABA increased gradually when the pretreatment duration was extended from 20 to 80 sec, and it reached a maximum value at the latter duration (Fig. 6). Analysis of the Intracellular Signal Transduction Route Involved in Suppression of IGABA by Caffeine. Considering the manner in which caffeine shifted the A B B 20 20 10 40 40 30 60 60 50 80 100 % % 0 0 1 1 2 10 100 1000 μM control control 500 pA 30 sec theophylline 10 μM theophylline caffeine 10 μM caffeine GABA 10 μM F i g. 3. Concentration – response graphs for IGABA without pretreat- ment with caffeine (1) and with such pretreatment (100 µM, 2). Each point represents the mean ± s.e.m. for 8–10 DRG neurons. All GABA-induced currents were normalized with respect to the response induced by 1.0 mM GABA applied alone. Р и с. 3. Графіки «концентрація–відповідь» для струму, опосе- редкованого рецепторами ГАМК (IGABA), без попередньої оброб- ки кофеїном (1) та після неї (100 мкМ; 2). washout washout F i g. 4. Comparison of the modulatory effects of caffeine and theophylline on GABA receptor-mediated currents (IGABA). A) Current traces showing the inhibitory effects of theophylline and caffeine in the same concentrations on IGABA (traces for each group are from the same DRG neuron). B) Diagram of the mean intensities of inhibition of IGABA exerted by caffeine (n = 9) and theophylline (n = 9; comparisons were performed in the same DRG neurons). Р и с. 4. Порівняння модуляторних впливів кофеїну та теофіліну на струми, опосередковані рецепторами ГАМК (IGABA). A control control diazepam 1 μM caffeine 10 μM + + diazepam 1 μM washout washout 500 pA 30 sec –30 –20 –10 0 10 20 30 F i g. 5. Modulatory effects of caffeine and diazepam on GABA receptor-mediated current (IGABA). A) Potentiation of IGABA by isolated action of diazepam and inhibition of this current by simultaneous diazepam and caffeine preapplication (traces are from the same DRG neuron). B) Diagram of the normalized changes in the IGABA amplitude induced by isolated action of diazepam and simultaneous preapplication of diazepam and caffeine (n = 9). Р и с. 5. Модуляторні впливи кофеїну та діазепаму на струми, опосередковані рецепторами ГАМК. NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2015.—T. 47, № 2 133 CAFFEINE SUPPRESSES GABA RECEPTOR-MEDIATED CURRENT concentration – response curve of IGABA downward markedly (Fig. 3), it was hypothesized that such suppression is noncompetitive and is caused by modulation of the GABAA receptor function via intracellular phosphorylation. To elucidate this question, H-8 (an inhibitor of protein kinase A) was added to the solution in the recording pipette for intracellular dialysis using the repatch technique. In the neurons treated with 50 µM H-8, inhibition of IGABA by caffeine was abolished almost completely in 5 min (P < 0.01, n = 7) (Fig. 7A). As is seen in Fig. 7B, when the pipette was filled with the normal internal solution, caffeine-induced suppression of IGABA was 43.9 ± 5.1%, on average (n = 7). At the same time, suppression of IGABA induced by caffeine was dramatically weaker 30 min after intracellular dialysis of the cell with the H-8-containing solution (8.5 ± 4.5%, P < 0.01). Considering that there was damage of the plasma membrane after the first patch- clamping, and dialysis of the cell interior with the fluid contained in the patch pipette could modify the cell function, we also compared IGABA between the first patch-clamping group and the repatch control group using micropipettes filled with the normal internal solution. No significant difference of IGABA for two groups was found (n = 3, data not shown). DISCUSSION The mechanisms of caffeine antagonizing actions of GABA and glycine have been studied by Uneyama et al. [3] on freshly isolated rat hippocampal neurons. In our research, we confirmed the concentration-dependent inhibition exerted by caffeine (0.01-100 µM) on IGABA (100 µM; IC50 = 10 µM) in rat DRG neurons. We found that this effect is stronger than that in hippocampal neurons (range of caffeine concentrations 10-10,000 µM, IC50 = 3.6 mM). The difference between results of our study on DRG neurons and Uneyama’s experiments on hippocampal neurons is the following. In the latter study, caffeine shifted the GABA concentration-response curve rightward and increased Kd without changing the maximum response. These results were interpreted in such a way that the inhibitory effect in hippocampal neurons was competitive. Intracellular application of BAPTA (a calcium ion chelator) and H-8 (a PKA inhibitor) did not affect depression of IGABA by caffeine in the above neurons. On the contrary, our study showed that caffeine in DRG neurons shifted the GABA B 20 20 20 10 10 40 40 50 60 40 60 80 30 30 50 0 0 0 500 pA sec A control control caffeine 10 μM normal internal solution H8 internal solution caffeine 10 μM caffein washout washout 30 sec F i g. 6. Dependence of inhibition of GABA receptor-mediated current IGABA (100 µM) on the duration of pre-application of caffeine (10 µM). Each point represents the mean ± s.e.m. of 6-9 neurons. Р и с. 6. Залежність пригнічення струму, опосередкованого рецепторами ГАМК (100 мкМ ГАМК), від тривалості попередньої аплікації 10 мкМ кофеїну. F i g. 7. Analysis of the intracellular signal transduction route involved in suppression of IGABA by caffeine A) Effect of intracellular dialysis with H-8 (50 µM)-containing solution abolishing the inhibitory effect of caffeine (10 µM) on IGABA. B) Diagram comparing the inhibitory effect of caffeine (10 µM) with the normal internal solution and the effect after repatch with intracellular dialysis by H-8-containing solution (n = 7, P < 0.001) Р и с. 7. Аналіз шляху передачі внутрішньоклітинних сигналів, який залучений в пригнічення кофеїном струму, опосередкова- ного рецепторами ГАМК (IGABA). % % NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2015.—T. 47, № 2134 J. Y. YANG, G. YANG, J. REN et al. concentration – response curve downward and de- creased the maximum response to 57%. These results provide reasons to believe that the effect of caffeine in DRG neurons is noncompetitive with respect to the GABAA receptor-Cl– channel complex. Intracellular dialysis with H-8 using the repatch techniques almost completely abolished caffeine-induced inhibition of IGABA. It has been reported that phosphorylation and dephosphorylation of the GABAA receptor-chloride channel complex are involved in modulation of the GABA-evoked responses [13]. Biochemical studies have suggested that phosphorylation of the GABAA receptors by various protein kinases inhibits the function of these receptor [14]. In agreement with our results, many studies also showed that cyclic AMP-dependent protein kinase directly phosphorylates GABAA receptors and decreases GABA-activated currents in DRG and spinal cord neurons [15, 16]. We concluded that caffeine-induced inhibition of the GABA response is mediated by the action of intracellular cAMP and GABAA receptor phosphorylation via intracellular phosphoinositi- des/PKA. The discrepancy between our results and Uneyama’s report may be related to different specimens and remains to be further investigated. Previous studies showed that the excitatory effect of caffeine on the nervous system is due to its antagonizing action on A2A receptors [17]. Recent reports , however, suggested that the caffeine intracellular pathways may be related to phosphorylation of DARPP-32 (dopamine- and cyclic AMP-regulated phosphoprotein with a molecular mass 32,000) [17-20]. As was mentioned above, the mechanisms underlying the effects of caffeine are variable. It was shown that methylxanthine alkaloids compete with caffeine in binding to benzodiazepine receptors [21]. In our study, we found that when DRG neurons were treated with diazepam prior to GABA, IGABA was enhanced, while simultaneous application of diazepam and caffeine resulted in inhibition on IGABA (Fig. 5). Caffeine increases the cAMP level via two routes; it antagonizes adenosine receptors [17, 22] and inhibits intracellular phosphodiesterase (PDE) [7, 8]. As to the former aspect, we found that the inhibitory effect of adenosine on IGABA in rat DRG neurons is mediated by subunit A1 adenosine receptors and not by A2A receptors [23]. Therefore, caffeine may antagonize A1 receptors. In our experiments, there was no activation of the adenosine system. Thus, we can infer that the effect of caffeine in this situation may be due mainly to increase in the intracellular cAMP level via inhibition of PDE. As is known, caffeine acts directly on the endoplasmic reticulum providing release of calcium and increase in the intracellular calcium concentration. These processes may, in principle, contribute to caffeine-induced suppression of IGABA [24, 25]. In our experiment, however, the effect of an increase in the intracellular calcium level could not be significant because EGTA was present in the intracellular solution. Thus, we found in our study that caffeine inhibits functioning of GABAA receptors in rat DRG neurons. If primary afferent depolarization is related to the function of GABAA receptors, caffeine might counteract presynaptic inhibitory action on primary afferents. Inhibition of PDE is the most probable route via which caffeine exerts its influence on the GABA inhibitory system. The inhibitory effect of caffeine on GABAA receptors might be a significant aspect on the activating influence of this agent on both the CNS and the peripheral nervous system. Acknowledgments. This work was supported by grants from the National Natural Science Foundation of China (Nos. 81371437, 81371223, and 30871006) and Research Fund for the Doctoral Program of Higher Education of China (No. 20122105110010). All experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and international ethical standards. All efforts were made to minimize the number of animals used and their suffering. The authors of this study, J. Y. Yang, G. Yang, J. Ren, J. Zhao, and Sh. Li, confirm that the research and publication of the results were not associated with any conflicts regarding commercial or financial relations, relations with organizations and/or individuals who may have been related to the study, and interrelations betveen co-authors of the article. Дж. І. Янг1, Г. Янг2, Дж. Рен3, Дж. Жао2, Ш. Лі2 КОФЕЇН ПРИГНІЧУЄ В ПЕРВИННИХ СЕНСОРНИХ НЕЙРОНАХ ЩУРА СТРУМ, ОПОСЕРЕДКОВАНИЙ РЕЦЕПТОРАМИ ГАМК, ЗАВДЯКИ ГАЛЬМУВАННЮ ВНУТРІШНЬОКЛІТИННОЇ ФОСФОДІЕСТЕРАЗИ 1 Лікарня «Дружба», Далянь (Китай). 2 Далянський медичний університет (Китай). 4 Центральна лікарня м. Джінань (Китай). NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2015.—T. 47, № 2 135 CAFFEINE SUPPRESSES GABA RECEPTOR-MEDIATED CURRENT Р е з ю м е Досліджували впливи кофеїну на струми, опосередкова- ні рецепторами ГАМК (IGABA), в гостроізольованих пер- винних нейронах щура; використовували методику петч- клемп у конфігурації «ціла клітина». Аплікації ГАМК (10–1000 мкМ) викликали струми вхідного напрямку, амп- літуда котрих була залежною від концентрації; струми де- монстрували яскраво виражену десенситизацію. Попе- редня обробка кофеїном (0.01–100 мкМ) призводила до неконкурентного пригнічення IGABA. Вплив кофеїну зміщу- вав вниз криву концентрація–відповідь для ГАМК порів- няно з контролем. Теофілін демонстрував подібні й навіть сильніші гальмівні впливи на IGABA. Ізольована аплікація 1 мкМ діазепаму посилювала IGABA, тоді як попередня обробка 10 мкМ кофеїну перед дією 1 мкМ діазепаму пригнічува- ла цей струм. Внутрішньоклітинна аплікація H-8 (інгібіто- ру протеїнкінази А) дуже значно послаблювала гальмівний вплив кофеїну на IGABA. Оскільки деполяризація терміна- лий первинних аферентів опосередковується рецептора- ми ГАМК, наші результати вказують на те, що кофеїн може протистояти пресинаптичному гальмуванню первинних афе- рентів. Ефекти кофеїну опосередковуються гальмуванням внутрішньоклітинної фосфодіестерази. 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Caffeine Suppresses GABA Receptor-Mediated Current in Rat Primary Sensory Neurons Via Inhibition of Intracellular Phosphodiesterase

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