Involvement of Src tyrosine kinase and mitogen-activated protein kinase in the facilitation of calcium channels in rat nucleus of the tractu
1 Department of Physiology, Tokyo Dental College, 1-2-2 Masago, Mihama-ku, Chiba 261-8502, Japan
Abstract
It is recognized that brain contains all the components of the renin–angiotensin systems (RAS). The nucleus of the tractus solitarius (NTS) is known to play a major role in the regulation of cardiovascular, respiratory, gustatory, hepatic and swallowing functions. Voltage-dependent Ca2+ channels (VDCCs) serve as crucial mediators of membrane excitability and Ca2+-dependent functions such as neurotransmitter release, enzyme activity and gene expression. The purpose of this study was to investigate the effects of angiotensin II (Ang II) on VDCC currents (ICa) in the NTS using patch-clamp recording methods. An application of Ang II caused facilitation of L-type ICa in a concentration-dependent manner with an EC50 of 167 nM and a Hill coefficient of 1.73. AT1 receptor antagonist losartan antagonized the Ang II-induced facilitation of ICa. Intracellular dialysis of the Gi-protein antibody attenuated the Ang II-induced facilitation of ICa. Both Src tyrosine kinase inhibitor and mitogen-activated protein kinase (MAPK) inhibitor attenuated the Ang II-induced facilitation of ICa. p38 MAPK inhibitor also attenuated the Ang II-induced facilitation of ICa. These results indicate that Ang II facilitates L-type VDCCs via Gi-proteins involving Src tyrosine kinase and p38 MAPK kinase mediated by AT1 receptors in NTS.
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Introduction
Angiotensin II (Ang II) has various physiological effects that are mediated by the brain, including stimulation of increased blood pressure, water and sodium intake, vasopressin secretion, and modulation of baroreflex function (Campagnole-Santos et al. 1988; Phillips & Sumners, 1998). Ang II also acts as a neurotransmitter in the central nervous system mediated by G-protein-coupled receptors (GPCRs, Bottari et al. 1993).
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The nucleus of the tractus solitarius (NTS) is known to play a major role in the regulation of cardiovascular, respiratory, gustatory, hepatic and swallowing functions (Lawrence & Jarrott, 1996; Jean, 2001). The NTS appears not to be a simple ‘relay’ nucleus, but performs complex integration of information from multiple synaptic inputs of both peripheral and central origins.
Voltage-dependent Ca2+ channels (VDCCs) serve as crucial mediators of membrane excitability and Ca2+-dependent functions such as neurotransmitter release, enzyme activity and gene expression. The modulation of VDCCs is believed to be an important means of regulating Ca2+ influx and thus has a direct influence on many Ca2+-dependent processes. Modulation of VDCCs by Ang II has been previously described in various types of cells. However, the effect of Ang II on VDCCs in NTS has not yet been clarified, and little is known about signal transduction pathways in NTS.
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Tyrosine phosphorylation is an important regulator of cell function (Schlessinger & Ullrich, 1992). Furthermore, increased tyrosine phosphorylation is associated with increased intracellular Ca2+ concentration ([Ca2+]i) during cell proliferation and migration. Although the mechanisms linking tyrosine phosphorylation to the changes in [Ca2+]i are not fully understood, in some cases increased opening of VDCCs has been proposed to underlie this effect (Hughes, 1995). Several studies have demonstrated that tyrosine kinase modulates VDCCs in a variety of cell types (Cataldi et al. 1996), suggesting that tyrosine phosphorylation may be a ubiquitous regulatory mechanism for VDCCs.
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Consequently, it is the purpose of this study to investigate the effects of Ang II on VDCC currents (ICa) in NTS.
Methods
Cell preparation
Experiments were conducted according to international guidelines on the use of animals for experimentation. Young Wistar rats (7–18 days old) were decapitated and their brains quickly removed and submerged in ice-cold artificial cerebrospinal fluid (aCSF) saturated with 95% O2 and 5% CO2 of the following composition (mM): NaCl 126, NaHCO3 26.2, NaH2PO4 1, KCl 3, MgSO4 1.5, CaCl2 1.5 and glucose 30; pH 7.4. Thin transverse slices from brainstems, 400 μm in thickness, were prepared by a tissue slicer (DTK-1000; Dosaka EM Co., Ltd, Kyoto, Japan). After being sectioned, 3–5 slices obtained from a single brain were transferred to a holding chamber and stored in oxygenated aCSF at room temperature for at least 40 min before use. Slices were then transferred to a conical tube containing gently bubbled aCSF at 36°C to which 1.8 U ml–1 dispase (grade I; 0.75 ml slice–1) was added. After 60 min incubation, slices were rinsed with enzyme-free aCSF. Under a dissecting microscope, the NTS region was micropunched and placed on a poly L-lysine-coated coverslip. The cells were then dissociated by trituration using progressively smaller diameter pipettes and allowed to settle on a coverslip for 20 min.
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Whole-cell patch-clamp recordings
Voltage-clamp recordings were conducted using the whole-cell configuration of the patch-clamp technique (Hamill et al. 1981). Fabricated recording pipettes (2–3 M) were filled with internal solution of the following composition (mM): 100 CsCl, 1 MgCl2, 10 Hepes, 10 BAPTA, 3.6 MgATP, 14 Tris2phosphocreatine (CP) and 0.1 GTP, plus 50 U ml–1 creatine phosphokinase (CPK). The pH was adjusted to 7.2 with CsOH. The inclusion of CP and CPK effectively reduced ‘rundown’ of ICa. After the formation of a giga seal, in order to record ICa, the extracellular solution was changed from Krebs solution to a solution containing the following (mM): 67 choline chloride, 100 tetraethylammonium chloride, 5.3 KCl, 5 CaCl2 and 10 Hepes. The pH was adjusted to 7.4 with Tris base. Command voltage protocols were generated with computer software pCLAMP version 8 (Axon Instruments, Union City, CA, USA) and transformed to an analog signal using a DigiData 1200 interface (Axon Instruments). The command pulses were applied to cells through an L/M-EPC7 amplifier (HEKA Elektronik, Lambrecht, Germany). The currents were recorded with the amplifier and computer software pCLAMP 8 acquisition system. Access resistance (< 15 M) was determined by transient responses to voltage commands. Access resistance compensation was not used. To ascertain that no major changes in the access resistance had occurred during the recordings a 5 mV, 10 ms pulses was used before ICa was evoked.
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Materials
Ang II was purchased from Peptide Institute (Japan). Losartan was a gift from Merck (Osaka, Japan). PD-123,319 ditrifluoroacetate was purchased from Research Biochemical International (Natick, MA, USA). Anti-Gi, anti-Gs, anti-Gq/11 and anti-Gantibodies were purchased from Upstate biotechnology (Lake Placid, NY, USA). All antibodies were from rabbits immunized with a synthetic peptide corresponding to the COOH-terminal sequence of the human Gi, Gs, Gq/11 and Gsubunits, respectively. -Conotoxin GVIA (-CgTx GVIA) and -agatoxin IVA (-Aga IVA) were purchased from Peptide Institute. Nifedipine (Nif) was purchased from Sigma. U-73122 was purchased from Wako Pure Chemical Industries (Osaka, Japan). LY294002 and PP2 were purchased from Calbiochem. Genistein, lavendustin A and PD98,059 were purchased from Sigma. U0126, SB202190 and SP600125 were purchased from Biomol.
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Most drugs were dissolved in distilled water. Nifedipine, U-73122, PP2, PD98,059, U0126, SB202190 and SP600125 were dissolved in DMSO to a final concentration of 10 mM as a stock solution. The final concentration of DMSO in extracellular solutions was < 0.01%, which had no effect on ICa.
Calculations
The dose–response relations for Ang II were calculated by a non-linear least-squares fit to the normalized facilitation in ICa amplitude using the Hill equation: I(X) = Imax/[1 + (EC50/X)n], where I(X) is the relative current facilitation from the baseline, Imax is maximal I(X), X is the concentration of Ang II, and n is the Hill coefficient.
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Analysis and statistics
All data analyses were performed using the pCLAMP 8.0 acquisition system. Values in text and figures are expressed as mean ± S.E.M. Statistical analysis was done using Student's t test for comparisons between pairs of groups and one-way analysis of variance (ANOVA) followed by Dunnett's test. Probability (P) values of less than 0.05 were considered significant.
Results
, http://www.100md.com Ang II-induced facilitation of ICa
An example of Ang II-induced facilitation of ICa is shown in Fig. 1. ICa was evoked every 20 s with a 100 ms depolarizing voltage step to 0 mV from a holding potential of –80 mV. As shown in Fig. 1A and B, application of 1 μM Ang II rapidly and reversibly facilitated ICa. When Ang II was applied twice to the same neuron, a rapid desensitization could be observed to its effects. While ICa facilitation by the first application of Ang II was 32.1%, ICa facilitation by the second application was only 9.1% in this neuron (Fig. 1B).
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A, typical superimposed ICa traces at the times indicated in the time course graph B. ICa were evoked from a holding potential of –80 mV by a 100 ms voltage step to 0 mV at 20 s intervals. B, typical time course of Ang II-induced facilitation of ICa. Ang II (1 μM) was bath-applied during the time indicated by the filled bar. C, current–voltage relations of ICa evoked by a series of voltage steps from a holding potential of –80 mV to test potentials between –80 and +40 mV in +10 mV increments in the absence () and presence () of 1 μM Ang II. Values of ICa are the averages of five neurons. D, bar graphs are means ± S.E.M. of ICa values before and after application of Ang II (1 μM). Data are from 12 neurons. *P < 0.05 compared with control, ANOVA. E, dose dependence of Ang II-induced facilitation of ICa. Numbers in parentheses indicate the number of neurons tested.
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The current–voltage relations measured before and during application of Ang II (1 μM) are shown in Fig. 1C. From a holding potential of –80 mV, ICa was activated after –30 mV with a peak current amplitude at 0 mV. Ang II did not shift the current–voltage relations (n = 5). Mean ICa values at 0 mV before and after application of 1 μM Ang II are shown in Fig. 1D.
The dose–response relations in the Ang II-induced facilitation of ICa are shown in Fig. 1E. Application of 1 nM to 100 μM Ang II rapidly and reversibly facilitated ICa in 135 of 169 neurons without changing current kinetics. For the generation of the concentration–response curve, Ang II concentrations were applied randomly, and not all concentrations were tested in a single neuron. Figure 1E shows that progressive increases in Ang II concentration resulted in progressively greater facilitation of ICa.
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Pharmacological studies have clearly identified two classes of Ang II receptors, i.e. AT1 and AT2 receptors.
To determine which receptor subtypes contribute to Ang II-induced facilitation of ICa in NTS, the effect of Ang II on ICa in neurons treated with specific antagonists was investigated. In these experiments, specific antagonists were applied prior to Ang II. Application of AT1 receptor antagonist losartan (1 μM for 3 min after assuming the whole-cell configuration) was without effect on basal ICa but blocked the Ang II-induced facilitation of ICa. However, the AT2 receptor antagonist PD-123,319 (1 μM for 3 min after assuming the whole-cell configuration) was without effect on Ang II-induced facilitation of ICa (32.2 ± 3.3% for Ang II only, 5.1 ± 1.6% for Ang II in neurons treated with losartan, and 28.2 ± 2.9% for Ang II in neurons treated with PD-123,319, n = 12, 6 and 5, respectively, Fig. 2A).
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A, antagonism of Ang II-induced facilitation of ICa. The histogram demonstrates the degree of ICa facilitation by 1 μM Ang II, Ang II after losartan (1 μM) and Ang II after PD-123,319 (1 μM). B, G-protein selectivity of Ang II-induced facilitation of ICa. The histogram demonstrates the degree of ICa facilitation by 1 μM Ang II in control (recording pipette was filled with GTP) and after intracellular dialysis with anti-Gi antibody, boiled anti-Gi antibody (90°C for 30 min), anti-Gs antibody, anti-Gq/11 antibody and anti-Gantibody. Numbers in parentheses indicate the number of neurons tested. *P < 0.05 compared with control, ANOVA.
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These results indicate that Ang II-induced facilitation of ICa was mediated by AT1 receptors in NTS.
Pharmacological characterization of G-protein subtypes and VDCC subtypes in Ang II-induced facilitation of ICa
To characterize the G-protein subtypes in Ang II-induced facilitation of ICa, specific antibodies raised against the Gi-, Gs-, Gq/11- and G-protein were used. Experiments were performed using pipette solutions containing each G-protein antibody. In these experiments, the G-protein antibody (1: 50 dilution; the final concentration was approximately 0.5 mg ml–1) was dissolved in the internal solution. The tip of the recording pipette was filled with the standard internal solution, and the pipette was then backfilled with solution which contained the G-protein antibody. In order to obtain the antibody effect, Ang II was applied 7 min after assuming the whole-cell configuration. Using the same protocol, we have demonstrated that intracellular dialysis of the Gq/11-protein antibody attenuated metabotropic glutamate receptor (mGluR) agonist-induced facilitation. mGluR-induced facilitation of ICa was unaffected by the antibody dialysis (Endoh, 2004).
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For the six neurons that were tested, intracellular dialysis of the Gi-protein antibody attenuated the Ang II-induced facilitation of ICa (11.2 ± 3.5%). In contrast, intracellular dialysis of Gs-, Gq/11- and G-protein antibodies did not attenuate the Ang II-induced facilitation of ICa (29.8 ± 3.5%, 30.3 ± 2.7% and 34.0 ± 2.6%, n = 7, 7 and 7, respectively). These results suggest that the Gi-proteins are involved in the Ang II-induced facilitation of ICa in NTS but Gs-, Gq/11- and G-proteins are not (Fig. 2B).
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Several electrophysiological studies have defined several pharmacologically distinct high-voltage-activated VDCCs on neuronal cell bodies, such as L-, N-, P-, Q- and R-type VDCCs. In this study, specific VDCC blockers were used to isolate each ICa component. Typical examples of sequential application of each selective blocker on ICa are shown in Fig. 3A and B. -CgTx GVIA, -Aga IVA and nifedipine (Nif) block N- (ICa-N), P/Q- (ICa-P/Q) and L-type (ICa-L) VDCCs, respectively. Mean percentages of ICa-N, ICa-P/Q, ICa-L and the resistance VDCC component (ICa-R) of total ICa were 26.2, 19.4, 39.8 and 15.9%, respectively (n = 4). All experiments were performed in the presence of 5.3 mM KCl in the external solution (see Methods). To ensure that all inward currents resulted from Ca2+ influx through VDCCs, i.e. to avoid the possibility of K+ influx, Cd2+ was applied after each selective VDCC blocker. As shown in Fig. 3A and B, all VDCC blockers and Cd2+ completely blocked ICa. Thus, it can be considered that the ICa obtained only resulted from Ca2+ influx.
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A, pharmacological characterization of four ICa components by sequential application of each VDCC blocker. Typical superimposed ICa traces at the times indicated in the time course graph B. B, typical time course of sequential application of each selective VDCC blocker on ICa. -CgTx GVIA, -Aga IVA, Nif and Cd2+ were bath applied during the time indicated by the filled bar. -CgTx GVIA, -Aga IVA, Nif and Cd2+ were bath-applied during the time indicated by each bar. C, histogram demonstrating the degree of ICa facilitation by 1 μM Ang II on L + R types (after treatment with -CgTx GVIA + -Aga IVA), N + R types (after treatment with Nif + -Aga IVA) and P/Q + R types (after treatment with Nif + -CgTx GVIA) VDCCs. Numbers in parentheses indicate the number of neurons tested. *P < 0.05 compared with L + R types, ANOVA.
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We then investigated which types of VDCCs were facilitated by Ang II. When Nif (10 μM) + -Aga IVA (1 μM) and Nif + -CgTx GVIA (1 μM) were applied first, the resistant ICa was not affected by a subsequent application of Ang II (4.1 ± 1.4 and 3.8 ± 1.2%, n = 5 and 5, respectively). On the other hand, when -CgTx GVIA + -Aga IVA were applied first, the resistant ICa was facilitated by a subsequent application of Ang II (19.5 ± 1.8%, n = 6, Fig. 3C).
Before Ang II application, mean ICa values of each of ICa-N, ICa-P/Q, ICa-L and ICa-R were 134 ± 62.5, 121 ± 41.5, 268.5 ± 81.5 and 82 ± 45.5 pA, respectively (n = 4). After application of Ang II, mean ICa values of each ICa-N, ICa-P/Q, ICa-L and ICa-R had increased to 140 ± 58.5, 127 ± 50, 504.5 ± 81 and 80 ± 42 pA, respectively (n = 5).
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These results demonstrated that Ang II facilitated L-type VDCCs, without significantly affecting N- and P/Q-type VDCCs in NTS.
As shown in Fig. 3C, Ang II-induced facilitations of each ICa component were smaller than the total ICa facilitation (34.9 ± 3.3% for Ang II only, 19.5 ± 1.8% for Ang II in neurons treated with -CgTx GVIA + -Aga IVA, n = 20 and 6, respectively). It can be considered that extracellular application of VDCC blockers required too much time for the full Ang II effects to appear. As shown in Fig. 3A, the time required to reach steady-state ICa by the sequential application of each blocker was much more than other experiments. Each VDCC blocker was applied for 2–3 min. Thus, it was difficult to prevent rundown, even though we had added CP and CPK in the recording pipette. Alternatively, it may be possible that the blocking of N- and P/Q-type VDCCs would alter the physiological facilitation of L-type VDCCs.
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Characterization of second messengers in Ang II-induced facilitation of ICa
Many studies have revealed that AT1 receptors are involved in either activation of phospholipase C (PLC) and stimulation of phosphoinositide (PI) hydrolysis or inhibition of adenylyl cyclase, depending on the cell or tissue type. Phosphatidylinositol-3 kinase (PI3K), which phosphorylates phosphatidylinositol-4,5-bisphosphate (PIP2) to phosphatidylinositol-3,4,5-trisphosphate (PIP3) (Auger et al. 1989), is known to be activated by Ang II. In vascular smooth muscle cells, Ang II is also known to activate several other kinases, such as tyrosine kinases (Marrero et al. 1995) and PI3K (Saward & Zahradka, 1997).
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To evaluate the possible contribution of PLC to the Ang II-induced facilitation of ICa, the effects of Ang II on ICa in neurons treated with U-73122 (which is a membrane-permeable aminosteroid that blocks the phosphatidylinositol-specific PLC) (Bleasdale et al. 1990) were investigated.
In order to avoid the effects of desensitization, each experiment was performed in individual neurons. Thus, Ang II-induced effects were not repeatable in the same neuron.
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In seven neurons tested, treatment with U-73122 (10 μM for 15 min before patch clamp experiments) did not attenuate the Ang II-induced facilitation of ICa (34.9 ± 3.3% for Ang II only, 35.0 ± 2.7% for Ang II in neurons treated with U-73122, n = 20 and 7, respectively, Figs 4A and B, and 8).
A, typical superimposed ICa traces at the times indicated in the time course of graph B. B, typical time course of Ang II-induced facilitation of ICa in a neuron treated with PLC inhibitor U-73122 (10 μM for 15 min before patch clamp experiments). C, histogram demonstrating the degree of ICa facilitation by 1 μM Ang II in control (untreated neurons), after 5, 10, 20 and 50 μM U-73122. Numbers in parentheses indicate the number of neurons tested. D, typical superimposed ICa traces at the times indicated in the time course graph E. E, typical time course of Ang II-induced facilitation of ICa in a neuron treated with PI3K inhibitor LY294002 (10 μM for 10 min before patch clamp experiments). Ang II (1 μM) was bath-applied during the time indicated by the filled bar.
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A, comparison of ICa values in various conditions in the presence of DMSO in the pipette solution. Histogram demonstrating the degree of ICa values before application of any drugs, application of 1 μM Ang II, genistein alone in the pipette solution, genistein in the pipette solution + 1 μM Ang II, lavendustin A alone in the pipette solution, lavendustin A in the pipette solution + 1 μM Ang II, PD98,059 alone in the pipette solution and PD98,059 in the pipette solution + 1 μM Ang II. B, histogram demonstrating the degree of ICa facilitation by 1 μM Ang II in control (untreated neurons), after U-73122, LY294002, genistein, lavendustin A, PP2, PD98,059, U0126, SB202190 and SP600125. Numbers in parentheses indicate the number of neurons tested. *P < 0.05 compared with control, ANOVA.
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In order to avoid sampling errors, 20 control neurons were used for comparison in the following experiments. These values were obtained in paired experiments, i.e. response to Ang II before and after drug treatment.
To ensure that U-73122 did not attenuate the Ang II-induced facilitation of ICa, other concentrations (5, 20 and 50 μM) of U-73122 were used. As shown in Fig. 4C, even these concentrations of U-73122 did not attenuate the Ang II-induced facilitation of ICa.
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To evaluate the possible contribution of PI3K to the Ang II-induced facilitation of ICa, the effects of Ang II on ICa in neurons treated with LY294002 (a selective PI3K inhibitor) were investigated. In seven neurons tested, treatment with LY294002 (10 μM for 10 min before patch clamp experiments) did not attenuate the Ang II-induced facilitation of ICa (34.9 ± 3.3% for Ang II only, 33.0 ± 2.9% for Ang II in neurons treated with LY294002, n = 20 and 7, respectively, Figs 4D and E, and 8).
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To evaluate the possible contribution of tyrosine kinases to the Ang II-induced facilitation of ICa, the effects of Ang II on ICa in neurons treated with genistein (a tyrosine kinase inhibitor) were investigated. In six neurons tested, treatment with genistein (10 μM for 2 min after assuming the whole-cell configuration) attenuated the Ang II-induced facilitation of ICa (34.9 ± 3.3% for Ang II only, 11.5 ± 2.7% for Ang II in neurons treated with genistein, n = 20 and 6, respectively, Fig. 5A and B). Similar results were obtained with a treatment of lavendustin A, which is also a tyrosine kinase inhibitor. In another six neurons tested, treatment with lavendustin A (10 μM for 2 min after assuming the whole-cell configuration) attenuated the Ang II-induced facilitation of ICa (34.9 ± 3.3% for Ang II only, 13.2 ± 3.2% for Ang II in neurons treated with lavendustin A, n = 20 and 6, respectively).
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A, typical superimposed ICa traces at the times indicated in the time course graph B. B, typical time course of Ang II-induced facilitation of ICa in a neuron treated with genistein (10 μM for 2 min). Genistein (10 μM) and Ang II (1 μM) were bath-applied during the time indicated by the open and filled bars, respectively. C, typical superimposed ICa traces at the times indicated in the time course graph D. D, typical time course of Ang II-induced facilitation of ICa in a neuron treated with PP2 (10 μM for 2 min). PP2 (10 μM) and Ang II (1 μM) were bath-applied during the time indicated by the open and filled bars, respectively. E, typical superimposed ICa traces at the times indicated in the time course graph F. F, typical time course of Ang II-induced facilitation of ICa in a neuron treated with PD98,059 (10 μM for 2 min). PD98,059 (10 μM) and Ang II (1 μM) were bath-applied during the time indicated by the open and filled bars, respectively.
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To evaluate the possible contribution of Src tyrosine kinases to the Ang II-induced facilitation of ICa, the effects of Ang II on ICa in neurons treated with PP2 (a Src tyrosine kinase inhibitor) were investigated. In seven neurons tested, treatment with PP2 (10 μM for 2 min after assuming the whole-cell configuration) attenuated the Ang II-induced facilitation of ICa (34.9 ± 3.3% for Ang II only, 11.2 ± 3.3% for Ang II in neurons treated with PP2, n = 20 and 7, respectively, Figs 5C and D, and 8).
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To evaluate the possible contribution of mitogen-activated protein kinase (MAPK) tyrosine kinases to the Ang II-induced facilitation of ICa, the effects of Ang II on ICa in neurons treated with PD98,059 (a MAPK tyrosine kinase inhibitor) were investigated. In another seven neurons tested, treatment with PD98,059 (10 μM for 2 min after assuming the whole-cell configuration) attenuated the Ang II-induced facilitation of ICa (34.9 ± 3.3% for Ang II only, 14.4 ± 3.1% for Ang II in neurons treated with PD98,059, n = 20 and 7, respectively, Figs 5E and F, and 8).
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It has been reported that genistein acts as a VDCC blocker (Belevych et al. 2002). In fact, genistein blocked ICa from –620 ± 182.5 pA to –501 ± 154 pA. Before application of genistein, the mean percentages of ICa-N, ICa-P/Q, ICa-L and resistance VDCC component (ICa-R) of total ICa were 26.2, 19.4, 39.8 and 15.9%, respectively (n = 4). In the presence of genistein (10 μM for 2 min in bath solution), mean percentages of ICa-N, ICa-P/Q, ICa-L and ICa-R of total ICa were 25.8, 18.5, 41.2 and 16.9%, respectively (n = 5). These results suggest that the abrogated Ang II effect cannot be explained by selective inhibition of ICa-L. Therefore, the effects of Ang II on ICa in neurons dialysed with a pipette solution containing genistein and lavendustin A were investigated. In each of six neurons tested, intracellular application of genistein (50 μM for 7 min after assuming the whole-cell configuration) and lavendustin A (50 μM for 7 min after assuming the whole-cell configuration) attenuated the Ang II-induced facilitation of ICa (31.0 ± 3.2% for Ang II only, 8.4 ± 2.7% for Ang II in neurons dialysed with genistein, 9.2 ± 3.2% for Ang II in neurons dialysed with lavendustin A, n = 5, 6 and 6, respectively, Figs 6A–D, and 8).
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A, typical superimposed ICa traces at the times indicated in the time course graph B. B, typical time course of Ang II-induced facilitation of ICa in a neuron dialysed with a pipette solution containing 50 μM genistein. C, typical superimposed ICa traces at the times indicated in the time course graph D. D, typical time course of Ang II-induced facilitation of ICa in a neuron dialysed with a pipette solution containing 50 μM lavendustin A. Ang II (1 μM) was bath-applied during the time indicated by the filled bar. E, typical superimposed ICa traces at the times indicated in the time course graph F. F, typical time course of Ang II-induced facilitation of ICa in a neuron dialysed with a pipette solution containing 50 μM PD98,059. Ang II (1 μM) was bath-applied during the time indicated by the filled bar.
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The effects of Ang II on ICa in neurons dialysed with a pipette solution containing PD98,059 were investigated. In seven neurons tested, intracellular application of PD98,059 (50 μM for 7 min after assuming the whole-cell configuration) attenuated the Ang II-induced facilitation of ICa (31.0 ± 3.2% for Ang II only, 7.3 ± 3.1% for Ang II in neurons dialysed with PD98,059, n = 5 and 7, respectively, Figs 6E and F, and 8).
These results indicate that Ang II facilitates VDCCs involving Src tyrosine kinase and MAPK in NTS.
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Characterization of MAPK subtypes in Ang II-induced facilitation of ICa
There are three major MAPK subgroups identified, including the extracellular signal-regulated kinases (ERK1/2), p38 MAPK and c-jun-NH2-terminal kinases (JNKs).
To evaluate the possible contribution of ERK1/2 to the Ang II-induced facilitation of ICa, the effects of Ang II on ICa in neurons treated with U0126 (ERK1/2 inhibitor) were investigated. U0126 is a specific inhibitor of MEK1/2 that can block the phosphorylation of p42/p44 MAPK induced by various stimuli (Favata et al. 1998). In six neurons tested, treatment with U0126 (10 μM for 10 min before patch clamp experiments) did not attenuate the Ang II-induced facilitation of ICa (34.9 ± 3.3% for Ang II only, 25.8 ± 2.9% for Ang II in neurons treated with U0126, n = 20 and 6, respectively, Figs 7A and B, and 8).
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A, typical superimposed ICa traces at the times indicated in the time course graph B. B, typical time course of Ang II-induced facilitation of ICa in a neuron treated with U0126 (10 μM for 10 min before patch clamp experiments). C, typical superimposed ICa traces at the times indicated in the time course graph D. D, typical time course of Ang II-induced facilitation of ICa in a neuron treated with SB202190 (10 μM for 10 min before patch clamp experiments). Ang II (1 μM) was bath-applied during the time indicated by the filled bar. E, typical superimposed ICa traces at the times indicated in the time course graph F. F, typical time course of Ang II-induced facilitation of ICa in a neuron treated with SP600125 (10 μM for 10 min). Ang II (1 μM) was bath-applied during the time indicated by the filled bar.
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To evaluate the possible contribution of p38 to the Ang II-induced facilitation of ICa, the effects of Ang II on ICa in neurons treated with SB202190 (p38 MAPK inhibitor) were investigated. The imidazole compound SB202190 is a specific inhibitor of p38 MAPK (Jiang et al. 1996). In seven neurons tested, treatment with SB202190 (10 μM for 10 min before patch clamp experiments) attenuated the Ang II-induced facilitation of ICa (34.9 ± 3.3% for Ang II only, 2.8 ± 1.2% for Ang II in neurons treated with SB202190, n = 20 and 7, respectively, Figs 7C and D, and 8).
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To evaluate the possible contribution of JNK to the Ang II-induced facilitation of ICa, the effects of Ang II on ICa in neurons treated with SP600125 (JNK inhibitor) were investigated. In six neurons tested, treatment with SP600125 (10 μM for 10 min before patch clamp experiments) did not attenuate the Ang II-induced facilitation of ICa (34.9 ± 3.3% for Ang II only, 25.4 ± 2.6% for Ang II in neurons treated with SP600125, n = 20 and 6, respectively, Figs 7E and F, and 8).
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These results indicate that Ang II facilitates VDCCs involving p38 MAPK in NTS.
Discussion
The present study investigated the effects of Ang II on ICa in NTS. While Wang et al. have demonstrated that NADPH oxidase-derived reactive oxygen species (ROS) are involved in Ang II-induced facilitation of L-type VDCCs in NTS (Wang et al. 2004), this is the first demonstration that Ang II facilitates L type VDCCs via Gi-proteins involving Src tyrosine kinase and p38 MAPK kinase mediated by AT1 receptors in NTS. It can be considered that Ang II stimulates Src tyrosine kinase, which in turn activates the p38 MAPK pathways, resulting in facilitation of VDCCs in NTS.
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Ang II modulation of NTS neurons
It is generally accepted that Ang II elicits neuronal excitation of NTS. Several electrophysiological recordings from NTS both in vivo (Hegarty et al. 1996) and in vitro (Barnes et al. 1988, 1991; Qu et al. 1996) described an excitatory effect of Ang II on NTS. Ang II-induced facilitation of VDCCs demonstrated in this study may lead to depolarization of NTS. On the other hand, we cannot rule out the fact that NTS expresses Ca2+-activated K+ (KCa) channels (Tell & Bradley, 1994), which can hyperpolarize the membrane (Halliwell, 1990; Hille, 2001). In fact, several studies in different cells show that Ang II first induces membrane hyperpolarization, before membrane depolarization (Quinn et al. 1987; Chorvatova et al. 1995). This stems from the fact that activation of the AT1 receptor induces facilitation of VDCCs which consequently increases permeability to K+ ions drawing the membrane potential to negative values. Increases in [Ca2+]i induced by Ang II precede this effect and lead to an initial hyperpolarization of the membrane. Afterwards, membrane depolarization occurs until a plateau is reached; then neurons repolarize towards control values. Furthermore, Ang II may very likely not exclusively facilitate VDCCs but also other G-protein-dependent channels. Ang II-induced activation of G-protein-gated inwardly rectifying K+ (GIRK) channels could also be candidates for hyperpolarization. In fact, it has been demonstrated that Ang II modulates several K+ channels (Pan et al. 2001), including GIRK channels (Hoyer et al. 1991) in other tissues. Thus, it will be important to clarify the mechanisms of integration of such G-protein signallings in the neuronal excitation.
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Wang et al. (2002) have demonstrated that application of 2 μM Ang II facilitates L-type VDCCs in NTS. Treatment with MnTBAP (the cell-permeant ROS scavenger) attenuated Ang II -induced facilitation of L-type VDCCs. In addition, treatment with apocynin (an inhibitor of NADPH oxidase assembly) and intracellular dialysis of gp91ds (NADPH oxidase peptide inhibitor) also attenuated Ang II-induced facilitation of L-type VDCCs. These results suggested that NADPH oxidase-derived ROS are involved in Ang II-induced facilitation of L-type VDCCs in NTS. These authors also suggested that such facilitation of L-type VDCCs provides an explanation for the finding that Ang II increases neuronal excitability and spontaneous activity (Ferguson et al. 2001).
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The effect of Ang II on NTS function
There are local renin–angiotensin systems (RASs) in regions such as the kidney and heart, and that dysfunction of these systems may lead to changes in the regulation of blood pressure. Recently, it has been thought likely that all components of the RAS are present in the brain, and local production of angiotensin peptides has been shown in several brain areas (Johnston et al. 1992; Bader & Ganten, 2002). Stimulation of brain RAS leads to increases in blood pressure, attenuation of the baroreceptor heart-rate reflex, stimulation of drinking, and the release of various hormones including vasopressin (Steckelings et al. 1992).
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Several demonstrations suggest that the NTS contains all of the components of RAS including angiotensinogen, angiotensin-converting enzyme (ACE), Ang II and Ang II receptors (Allen et al. 1998). The homeostatic functions of Ang II are mediated, in part, through AT1 receptors in the NTS, which are components of the sensory vagal complex that responds to baroreceptor and volume receptor information by ascending projections to brain regions (Fitzsimons, 1998).
Some reports demonstrated that NTS contributes to baroreceptor regulation. However, the effect of Ang II on the NTS to modulate baroreceptor regulation of blood pressure is complex. Microinjection of Ang II into the NTS has been reported to yield not only pressor, but also depressor effects (Rettig et al. 1986; Mosqueda-Garcia et al. 1990; Fow et al. 1994). Microinjection of low doses of Ang II (200 fmol) into the NTS produced hypotension and bradycardia that mimicked stimulation of the baroreceptor reflex (Fow et al. 1994). Furthermore, high doses of Ang II (1 pmol) microinjected into the NTS increased arterial pressure (Casto & Phillips, 1984; Rettig et al. 1986). The exact mechanisms by which Ang II produces pressor or depressor responses in the NTS are unknown. The activation of different receptor subtypes and affinities may be due to opposite effects of this peptide (Andresen, 1994; Fow et al. 1994). Alternatively, it can be considered that neuronal networks exist within the NTS. NTS neurons can be divided into two groups, GABAergic and glutamatergic (Mifflin & Felder, 1990; Brooks et al. 1992). As mentioned above, microinjection of Ang II into NTS reduces baroreflex function. It can be considered that facilitation of VDCCs on GABAergic neurons can enhance GABA release and therefore a reduced baroreflex function. Wong et al. (2002) proposed an enhancement of GABA release induced by NO that was generated by Ca2+ activation of NOS. The increased Ca2+ influx through L-type VDCCs could activate the same mechanism. However, in the case of glutamatergic neurons, it would have the opposite effect. In this study, it is not possible to identify distinct NTS subgroups.
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Ang II-induced facilitation of VDCCs involving Src
Protein tyrosine kinases (PTKs) are classified into two major groups: receptor-linked PTKs (RTKs) and non-receptor-linked PTKs (non-RTKs). RTKs, such as receptors for growth factor (e.g. platelet-derived growth factor (PDGF), or epidermal growth factor (EGF)), are attached to the receptor as a special domain. Interaction of a specific ligand with the receptor results in dimerization of the receptor, auto-phosphorylation of specific tyrosine residues, and expression of tyrosine kinase activity for specific substrates. In the other class, the non-RTKs such as Pyk, FAK, JAK, Src and products of Abl and Fes are cytoplasmic enzymes.
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Ang II-induced facilitation of VDCCs suggests that a principal effect of Ang II modulation might be regulation of Ca2+-dependent enzymes, in particular, transcription factors (Alberini et al. 1994). L-type VDCCs have been linked to not only somal action potentials, but also to the activation of Src, the modulation of RTK (Finkbeiner & Greenberg, 1996), and gene expression, including synthesis of ion channels (Murphy et al. 1991; Misra et al. 1994). During differentiation, L-type VDCC-mediated Ca2+ influx is essential for neurite outgrowth, synapse formation, survival and the shift to the mature action potential profile (Spitzer, 1994). Developmentally, Ca2+ influx would be expected to trigger influx-dependent cellular differentiation (Spitzer, 1994; Gu & Spitzer, 1995) and, because early action potential activity has been linked to establishing neuronal circuits (Katz & Shatz, 1996), potentially could contribute to pattern information. These studies strongly imply that Ang II-induced facilitation of L-type VDCCs would lead to a substantial increase in Ca2+ influx. Whether occurring as a result of spontaneous activity or mature action potential firing, such increases in [Ca2+]i may dramatically alter neuronal function.
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As shown in Fig. 5B, D and F, even under basal conditions, ICa could be attenuated by tyrosine kinase inhibitors. Precisely how Src kinase modulates the VDCCs is uncertain. Single channel studies in inside-out patches isolated from rat portal vein cells have demonstrated that genistein reduces the open probability of L-type VDCCs without altering unit amplitude or slope conductance (Liu & Sperelakis, 1997). A similar effect of genistein on steady-state inactivation of ICa has also been reported in other cell types (Yokoshiki et al. 1995; Liu et al. 1997). In addition, Fitzgerald & Dolphin (1997) have suggested that Src kinase can influence the gating of VDCCs. These data could be taken to implicate Src tyrosine kinase and/or MAPK kinase in modulating the inactivation process of VDCCs and imply a possible role for tyrosine phosphorylation/dephosphorylation in VDCC inactivation. Therefore, it can be considered that this kinase may represent a ubiquitous modulator of ion channels in many cell types.
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There is evidence for c-src being responsible for the modulation of L-type VDCCs in vascular smooth muscle, particularly when taken in conjunction with previous observations which have shown that VDCCs are facilitated by intracellular application of purified c-src (Wijetunge & Hughes, 1995), or a peptide that activates endogenous c-src (Wijetunge & Hughes, 1996). The idea that c-src may regulate L-type VDCCs receives further support from the observation that c-src can be found in immunoprecipitates of the 1c subunit of L-type VDCCs (Hu et al. 1998). Hence there is both functional and biochemical evidence closely linking c-src to the pore-forming 1c subunit of L-type VDCCs.
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Furthermore, L-type VDCCs have been implicated in the synaptic control of nuclear gene expression. Ang II has been shown to increase the expression of inducible transcription factors mediated by AT1 receptor-dependent mechanisms in the brain regions (Moellenhoff et al. 1998). The possibility exists that VDCCs play a role in regulating gene expression in these situations, thus serving as a focal point for Ang II-induced regulation of both neuronal excitability and protein expression. The idea that Src tyrosine kinase may regulate L-type VDCCs receives further support from the observation that Src tyrosine kinase can be found in immunoprecipitates of the 1c subunit of L-type VDCCs (Hu et al. 1998).
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In this study the EC50 was 167 nM, and the Hill slope coefficient was 1.7 in NTS. EC50 values for Ang II-induced facilitation of ICa have been calculated in other tissues. In rabbit ventricular myocytes (0.75 nM, Ichiyanagi et al. 2002) and rat heart myocytes (0.6 nM, Allen et al. 1988) it was found to be 100-fold more potent than in NTS. The discrepancy observed in these studies carried out in different cell types may be due to cell-dependent differences in the regulation of membrane excitability.
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As shown in Fig. 8, genistein and PP2 failed to completely block the response of Ang II. What other kinases/mechanisms are involved in the regulation of the opening of VDCCsIt has been reported that arachidonic acid (AA) is a second messenger in the regulation of VDCCs. Several GPCRs can activate phospholipase A (PLA), which in turn catalyses the release of AA (Axelrod et al. 1988). AA-induced facilitation of VDCCs has been reported (Vacher et al. 1989; Huang et al. 1992; Chesnoy-Marchais & Fritsch, 1994). Ang II-induced AA release has been demonstrated recently (Li & Malik, 2005). Calcium–calmodulin protein kinase II (CaMKII) may also have a role in the AA-induced facilitation of VDCCs: it has been reported that Ang II-induced inhibition of Kv channel current involved CaMKII (Sun et al. 2003). Therefore, the Ang II receptor pathway should be investigated in a further study.
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Abstract
It is recognized that brain contains all the components of the renin–angiotensin systems (RAS). The nucleus of the tractus solitarius (NTS) is known to play a major role in the regulation of cardiovascular, respiratory, gustatory, hepatic and swallowing functions. Voltage-dependent Ca2+ channels (VDCCs) serve as crucial mediators of membrane excitability and Ca2+-dependent functions such as neurotransmitter release, enzyme activity and gene expression. The purpose of this study was to investigate the effects of angiotensin II (Ang II) on VDCC currents (ICa) in the NTS using patch-clamp recording methods. An application of Ang II caused facilitation of L-type ICa in a concentration-dependent manner with an EC50 of 167 nM and a Hill coefficient of 1.73. AT1 receptor antagonist losartan antagonized the Ang II-induced facilitation of ICa. Intracellular dialysis of the Gi-protein antibody attenuated the Ang II-induced facilitation of ICa. Both Src tyrosine kinase inhibitor and mitogen-activated protein kinase (MAPK) inhibitor attenuated the Ang II-induced facilitation of ICa. p38 MAPK inhibitor also attenuated the Ang II-induced facilitation of ICa. These results indicate that Ang II facilitates L-type VDCCs via Gi-proteins involving Src tyrosine kinase and p38 MAPK kinase mediated by AT1 receptors in NTS.
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Introduction
Angiotensin II (Ang II) has various physiological effects that are mediated by the brain, including stimulation of increased blood pressure, water and sodium intake, vasopressin secretion, and modulation of baroreflex function (Campagnole-Santos et al. 1988; Phillips & Sumners, 1998). Ang II also acts as a neurotransmitter in the central nervous system mediated by G-protein-coupled receptors (GPCRs, Bottari et al. 1993).
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The nucleus of the tractus solitarius (NTS) is known to play a major role in the regulation of cardiovascular, respiratory, gustatory, hepatic and swallowing functions (Lawrence & Jarrott, 1996; Jean, 2001). The NTS appears not to be a simple ‘relay’ nucleus, but performs complex integration of information from multiple synaptic inputs of both peripheral and central origins.
Voltage-dependent Ca2+ channels (VDCCs) serve as crucial mediators of membrane excitability and Ca2+-dependent functions such as neurotransmitter release, enzyme activity and gene expression. The modulation of VDCCs is believed to be an important means of regulating Ca2+ influx and thus has a direct influence on many Ca2+-dependent processes. Modulation of VDCCs by Ang II has been previously described in various types of cells. However, the effect of Ang II on VDCCs in NTS has not yet been clarified, and little is known about signal transduction pathways in NTS.
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Tyrosine phosphorylation is an important regulator of cell function (Schlessinger & Ullrich, 1992). Furthermore, increased tyrosine phosphorylation is associated with increased intracellular Ca2+ concentration ([Ca2+]i) during cell proliferation and migration. Although the mechanisms linking tyrosine phosphorylation to the changes in [Ca2+]i are not fully understood, in some cases increased opening of VDCCs has been proposed to underlie this effect (Hughes, 1995). Several studies have demonstrated that tyrosine kinase modulates VDCCs in a variety of cell types (Cataldi et al. 1996), suggesting that tyrosine phosphorylation may be a ubiquitous regulatory mechanism for VDCCs.
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Consequently, it is the purpose of this study to investigate the effects of Ang II on VDCC currents (ICa) in NTS.
Methods
Cell preparation
Experiments were conducted according to international guidelines on the use of animals for experimentation. Young Wistar rats (7–18 days old) were decapitated and their brains quickly removed and submerged in ice-cold artificial cerebrospinal fluid (aCSF) saturated with 95% O2 and 5% CO2 of the following composition (mM): NaCl 126, NaHCO3 26.2, NaH2PO4 1, KCl 3, MgSO4 1.5, CaCl2 1.5 and glucose 30; pH 7.4. Thin transverse slices from brainstems, 400 μm in thickness, were prepared by a tissue slicer (DTK-1000; Dosaka EM Co., Ltd, Kyoto, Japan). After being sectioned, 3–5 slices obtained from a single brain were transferred to a holding chamber and stored in oxygenated aCSF at room temperature for at least 40 min before use. Slices were then transferred to a conical tube containing gently bubbled aCSF at 36°C to which 1.8 U ml–1 dispase (grade I; 0.75 ml slice–1) was added. After 60 min incubation, slices were rinsed with enzyme-free aCSF. Under a dissecting microscope, the NTS region was micropunched and placed on a poly L-lysine-coated coverslip. The cells were then dissociated by trituration using progressively smaller diameter pipettes and allowed to settle on a coverslip for 20 min.
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Whole-cell patch-clamp recordings
Voltage-clamp recordings were conducted using the whole-cell configuration of the patch-clamp technique (Hamill et al. 1981). Fabricated recording pipettes (2–3 M) were filled with internal solution of the following composition (mM): 100 CsCl, 1 MgCl2, 10 Hepes, 10 BAPTA, 3.6 MgATP, 14 Tris2phosphocreatine (CP) and 0.1 GTP, plus 50 U ml–1 creatine phosphokinase (CPK). The pH was adjusted to 7.2 with CsOH. The inclusion of CP and CPK effectively reduced ‘rundown’ of ICa. After the formation of a giga seal, in order to record ICa, the extracellular solution was changed from Krebs solution to a solution containing the following (mM): 67 choline chloride, 100 tetraethylammonium chloride, 5.3 KCl, 5 CaCl2 and 10 Hepes. The pH was adjusted to 7.4 with Tris base. Command voltage protocols were generated with computer software pCLAMP version 8 (Axon Instruments, Union City, CA, USA) and transformed to an analog signal using a DigiData 1200 interface (Axon Instruments). The command pulses were applied to cells through an L/M-EPC7 amplifier (HEKA Elektronik, Lambrecht, Germany). The currents were recorded with the amplifier and computer software pCLAMP 8 acquisition system. Access resistance (< 15 M) was determined by transient responses to voltage commands. Access resistance compensation was not used. To ascertain that no major changes in the access resistance had occurred during the recordings a 5 mV, 10 ms pulses was used before ICa was evoked.
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Materials
Ang II was purchased from Peptide Institute (Japan). Losartan was a gift from Merck (Osaka, Japan). PD-123,319 ditrifluoroacetate was purchased from Research Biochemical International (Natick, MA, USA). Anti-Gi, anti-Gs, anti-Gq/11 and anti-Gantibodies were purchased from Upstate biotechnology (Lake Placid, NY, USA). All antibodies were from rabbits immunized with a synthetic peptide corresponding to the COOH-terminal sequence of the human Gi, Gs, Gq/11 and Gsubunits, respectively. -Conotoxin GVIA (-CgTx GVIA) and -agatoxin IVA (-Aga IVA) were purchased from Peptide Institute. Nifedipine (Nif) was purchased from Sigma. U-73122 was purchased from Wako Pure Chemical Industries (Osaka, Japan). LY294002 and PP2 were purchased from Calbiochem. Genistein, lavendustin A and PD98,059 were purchased from Sigma. U0126, SB202190 and SP600125 were purchased from Biomol.
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Most drugs were dissolved in distilled water. Nifedipine, U-73122, PP2, PD98,059, U0126, SB202190 and SP600125 were dissolved in DMSO to a final concentration of 10 mM as a stock solution. The final concentration of DMSO in extracellular solutions was < 0.01%, which had no effect on ICa.
Calculations
The dose–response relations for Ang II were calculated by a non-linear least-squares fit to the normalized facilitation in ICa amplitude using the Hill equation: I(X) = Imax/[1 + (EC50/X)n], where I(X) is the relative current facilitation from the baseline, Imax is maximal I(X), X is the concentration of Ang II, and n is the Hill coefficient.
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Analysis and statistics
All data analyses were performed using the pCLAMP 8.0 acquisition system. Values in text and figures are expressed as mean ± S.E.M. Statistical analysis was done using Student's t test for comparisons between pairs of groups and one-way analysis of variance (ANOVA) followed by Dunnett's test. Probability (P) values of less than 0.05 were considered significant.
Results
, http://www.100md.com Ang II-induced facilitation of ICa
An example of Ang II-induced facilitation of ICa is shown in Fig. 1. ICa was evoked every 20 s with a 100 ms depolarizing voltage step to 0 mV from a holding potential of –80 mV. As shown in Fig. 1A and B, application of 1 μM Ang II rapidly and reversibly facilitated ICa. When Ang II was applied twice to the same neuron, a rapid desensitization could be observed to its effects. While ICa facilitation by the first application of Ang II was 32.1%, ICa facilitation by the second application was only 9.1% in this neuron (Fig. 1B).
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A, typical superimposed ICa traces at the times indicated in the time course graph B. ICa were evoked from a holding potential of –80 mV by a 100 ms voltage step to 0 mV at 20 s intervals. B, typical time course of Ang II-induced facilitation of ICa. Ang II (1 μM) was bath-applied during the time indicated by the filled bar. C, current–voltage relations of ICa evoked by a series of voltage steps from a holding potential of –80 mV to test potentials between –80 and +40 mV in +10 mV increments in the absence () and presence () of 1 μM Ang II. Values of ICa are the averages of five neurons. D, bar graphs are means ± S.E.M. of ICa values before and after application of Ang II (1 μM). Data are from 12 neurons. *P < 0.05 compared with control, ANOVA. E, dose dependence of Ang II-induced facilitation of ICa. Numbers in parentheses indicate the number of neurons tested.
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The current–voltage relations measured before and during application of Ang II (1 μM) are shown in Fig. 1C. From a holding potential of –80 mV, ICa was activated after –30 mV with a peak current amplitude at 0 mV. Ang II did not shift the current–voltage relations (n = 5). Mean ICa values at 0 mV before and after application of 1 μM Ang II are shown in Fig. 1D.
The dose–response relations in the Ang II-induced facilitation of ICa are shown in Fig. 1E. Application of 1 nM to 100 μM Ang II rapidly and reversibly facilitated ICa in 135 of 169 neurons without changing current kinetics. For the generation of the concentration–response curve, Ang II concentrations were applied randomly, and not all concentrations were tested in a single neuron. Figure 1E shows that progressive increases in Ang II concentration resulted in progressively greater facilitation of ICa.
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Pharmacological studies have clearly identified two classes of Ang II receptors, i.e. AT1 and AT2 receptors.
To determine which receptor subtypes contribute to Ang II-induced facilitation of ICa in NTS, the effect of Ang II on ICa in neurons treated with specific antagonists was investigated. In these experiments, specific antagonists were applied prior to Ang II. Application of AT1 receptor antagonist losartan (1 μM for 3 min after assuming the whole-cell configuration) was without effect on basal ICa but blocked the Ang II-induced facilitation of ICa. However, the AT2 receptor antagonist PD-123,319 (1 μM for 3 min after assuming the whole-cell configuration) was without effect on Ang II-induced facilitation of ICa (32.2 ± 3.3% for Ang II only, 5.1 ± 1.6% for Ang II in neurons treated with losartan, and 28.2 ± 2.9% for Ang II in neurons treated with PD-123,319, n = 12, 6 and 5, respectively, Fig. 2A).
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A, antagonism of Ang II-induced facilitation of ICa. The histogram demonstrates the degree of ICa facilitation by 1 μM Ang II, Ang II after losartan (1 μM) and Ang II after PD-123,319 (1 μM). B, G-protein selectivity of Ang II-induced facilitation of ICa. The histogram demonstrates the degree of ICa facilitation by 1 μM Ang II in control (recording pipette was filled with GTP) and after intracellular dialysis with anti-Gi antibody, boiled anti-Gi antibody (90°C for 30 min), anti-Gs antibody, anti-Gq/11 antibody and anti-Gantibody. Numbers in parentheses indicate the number of neurons tested. *P < 0.05 compared with control, ANOVA.
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These results indicate that Ang II-induced facilitation of ICa was mediated by AT1 receptors in NTS.
Pharmacological characterization of G-protein subtypes and VDCC subtypes in Ang II-induced facilitation of ICa
To characterize the G-protein subtypes in Ang II-induced facilitation of ICa, specific antibodies raised against the Gi-, Gs-, Gq/11- and G-protein were used. Experiments were performed using pipette solutions containing each G-protein antibody. In these experiments, the G-protein antibody (1: 50 dilution; the final concentration was approximately 0.5 mg ml–1) was dissolved in the internal solution. The tip of the recording pipette was filled with the standard internal solution, and the pipette was then backfilled with solution which contained the G-protein antibody. In order to obtain the antibody effect, Ang II was applied 7 min after assuming the whole-cell configuration. Using the same protocol, we have demonstrated that intracellular dialysis of the Gq/11-protein antibody attenuated metabotropic glutamate receptor (mGluR) agonist-induced facilitation. mGluR-induced facilitation of ICa was unaffected by the antibody dialysis (Endoh, 2004).
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For the six neurons that were tested, intracellular dialysis of the Gi-protein antibody attenuated the Ang II-induced facilitation of ICa (11.2 ± 3.5%). In contrast, intracellular dialysis of Gs-, Gq/11- and G-protein antibodies did not attenuate the Ang II-induced facilitation of ICa (29.8 ± 3.5%, 30.3 ± 2.7% and 34.0 ± 2.6%, n = 7, 7 and 7, respectively). These results suggest that the Gi-proteins are involved in the Ang II-induced facilitation of ICa in NTS but Gs-, Gq/11- and G-proteins are not (Fig. 2B).
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Several electrophysiological studies have defined several pharmacologically distinct high-voltage-activated VDCCs on neuronal cell bodies, such as L-, N-, P-, Q- and R-type VDCCs. In this study, specific VDCC blockers were used to isolate each ICa component. Typical examples of sequential application of each selective blocker on ICa are shown in Fig. 3A and B. -CgTx GVIA, -Aga IVA and nifedipine (Nif) block N- (ICa-N), P/Q- (ICa-P/Q) and L-type (ICa-L) VDCCs, respectively. Mean percentages of ICa-N, ICa-P/Q, ICa-L and the resistance VDCC component (ICa-R) of total ICa were 26.2, 19.4, 39.8 and 15.9%, respectively (n = 4). All experiments were performed in the presence of 5.3 mM KCl in the external solution (see Methods). To ensure that all inward currents resulted from Ca2+ influx through VDCCs, i.e. to avoid the possibility of K+ influx, Cd2+ was applied after each selective VDCC blocker. As shown in Fig. 3A and B, all VDCC blockers and Cd2+ completely blocked ICa. Thus, it can be considered that the ICa obtained only resulted from Ca2+ influx.
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A, pharmacological characterization of four ICa components by sequential application of each VDCC blocker. Typical superimposed ICa traces at the times indicated in the time course graph B. B, typical time course of sequential application of each selective VDCC blocker on ICa. -CgTx GVIA, -Aga IVA, Nif and Cd2+ were bath applied during the time indicated by the filled bar. -CgTx GVIA, -Aga IVA, Nif and Cd2+ were bath-applied during the time indicated by each bar. C, histogram demonstrating the degree of ICa facilitation by 1 μM Ang II on L + R types (after treatment with -CgTx GVIA + -Aga IVA), N + R types (after treatment with Nif + -Aga IVA) and P/Q + R types (after treatment with Nif + -CgTx GVIA) VDCCs. Numbers in parentheses indicate the number of neurons tested. *P < 0.05 compared with L + R types, ANOVA.
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We then investigated which types of VDCCs were facilitated by Ang II. When Nif (10 μM) + -Aga IVA (1 μM) and Nif + -CgTx GVIA (1 μM) were applied first, the resistant ICa was not affected by a subsequent application of Ang II (4.1 ± 1.4 and 3.8 ± 1.2%, n = 5 and 5, respectively). On the other hand, when -CgTx GVIA + -Aga IVA were applied first, the resistant ICa was facilitated by a subsequent application of Ang II (19.5 ± 1.8%, n = 6, Fig. 3C).
Before Ang II application, mean ICa values of each of ICa-N, ICa-P/Q, ICa-L and ICa-R were 134 ± 62.5, 121 ± 41.5, 268.5 ± 81.5 and 82 ± 45.5 pA, respectively (n = 4). After application of Ang II, mean ICa values of each ICa-N, ICa-P/Q, ICa-L and ICa-R had increased to 140 ± 58.5, 127 ± 50, 504.5 ± 81 and 80 ± 42 pA, respectively (n = 5).
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These results demonstrated that Ang II facilitated L-type VDCCs, without significantly affecting N- and P/Q-type VDCCs in NTS.
As shown in Fig. 3C, Ang II-induced facilitations of each ICa component were smaller than the total ICa facilitation (34.9 ± 3.3% for Ang II only, 19.5 ± 1.8% for Ang II in neurons treated with -CgTx GVIA + -Aga IVA, n = 20 and 6, respectively). It can be considered that extracellular application of VDCC blockers required too much time for the full Ang II effects to appear. As shown in Fig. 3A, the time required to reach steady-state ICa by the sequential application of each blocker was much more than other experiments. Each VDCC blocker was applied for 2–3 min. Thus, it was difficult to prevent rundown, even though we had added CP and CPK in the recording pipette. Alternatively, it may be possible that the blocking of N- and P/Q-type VDCCs would alter the physiological facilitation of L-type VDCCs.
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Characterization of second messengers in Ang II-induced facilitation of ICa
Many studies have revealed that AT1 receptors are involved in either activation of phospholipase C (PLC) and stimulation of phosphoinositide (PI) hydrolysis or inhibition of adenylyl cyclase, depending on the cell or tissue type. Phosphatidylinositol-3 kinase (PI3K), which phosphorylates phosphatidylinositol-4,5-bisphosphate (PIP2) to phosphatidylinositol-3,4,5-trisphosphate (PIP3) (Auger et al. 1989), is known to be activated by Ang II. In vascular smooth muscle cells, Ang II is also known to activate several other kinases, such as tyrosine kinases (Marrero et al. 1995) and PI3K (Saward & Zahradka, 1997).
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To evaluate the possible contribution of PLC to the Ang II-induced facilitation of ICa, the effects of Ang II on ICa in neurons treated with U-73122 (which is a membrane-permeable aminosteroid that blocks the phosphatidylinositol-specific PLC) (Bleasdale et al. 1990) were investigated.
In order to avoid the effects of desensitization, each experiment was performed in individual neurons. Thus, Ang II-induced effects were not repeatable in the same neuron.
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In seven neurons tested, treatment with U-73122 (10 μM for 15 min before patch clamp experiments) did not attenuate the Ang II-induced facilitation of ICa (34.9 ± 3.3% for Ang II only, 35.0 ± 2.7% for Ang II in neurons treated with U-73122, n = 20 and 7, respectively, Figs 4A and B, and 8).
A, typical superimposed ICa traces at the times indicated in the time course of graph B. B, typical time course of Ang II-induced facilitation of ICa in a neuron treated with PLC inhibitor U-73122 (10 μM for 15 min before patch clamp experiments). C, histogram demonstrating the degree of ICa facilitation by 1 μM Ang II in control (untreated neurons), after 5, 10, 20 and 50 μM U-73122. Numbers in parentheses indicate the number of neurons tested. D, typical superimposed ICa traces at the times indicated in the time course graph E. E, typical time course of Ang II-induced facilitation of ICa in a neuron treated with PI3K inhibitor LY294002 (10 μM for 10 min before patch clamp experiments). Ang II (1 μM) was bath-applied during the time indicated by the filled bar.
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A, comparison of ICa values in various conditions in the presence of DMSO in the pipette solution. Histogram demonstrating the degree of ICa values before application of any drugs, application of 1 μM Ang II, genistein alone in the pipette solution, genistein in the pipette solution + 1 μM Ang II, lavendustin A alone in the pipette solution, lavendustin A in the pipette solution + 1 μM Ang II, PD98,059 alone in the pipette solution and PD98,059 in the pipette solution + 1 μM Ang II. B, histogram demonstrating the degree of ICa facilitation by 1 μM Ang II in control (untreated neurons), after U-73122, LY294002, genistein, lavendustin A, PP2, PD98,059, U0126, SB202190 and SP600125. Numbers in parentheses indicate the number of neurons tested. *P < 0.05 compared with control, ANOVA.
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In order to avoid sampling errors, 20 control neurons were used for comparison in the following experiments. These values were obtained in paired experiments, i.e. response to Ang II before and after drug treatment.
To ensure that U-73122 did not attenuate the Ang II-induced facilitation of ICa, other concentrations (5, 20 and 50 μM) of U-73122 were used. As shown in Fig. 4C, even these concentrations of U-73122 did not attenuate the Ang II-induced facilitation of ICa.
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To evaluate the possible contribution of PI3K to the Ang II-induced facilitation of ICa, the effects of Ang II on ICa in neurons treated with LY294002 (a selective PI3K inhibitor) were investigated. In seven neurons tested, treatment with LY294002 (10 μM for 10 min before patch clamp experiments) did not attenuate the Ang II-induced facilitation of ICa (34.9 ± 3.3% for Ang II only, 33.0 ± 2.9% for Ang II in neurons treated with LY294002, n = 20 and 7, respectively, Figs 4D and E, and 8).
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To evaluate the possible contribution of tyrosine kinases to the Ang II-induced facilitation of ICa, the effects of Ang II on ICa in neurons treated with genistein (a tyrosine kinase inhibitor) were investigated. In six neurons tested, treatment with genistein (10 μM for 2 min after assuming the whole-cell configuration) attenuated the Ang II-induced facilitation of ICa (34.9 ± 3.3% for Ang II only, 11.5 ± 2.7% for Ang II in neurons treated with genistein, n = 20 and 6, respectively, Fig. 5A and B). Similar results were obtained with a treatment of lavendustin A, which is also a tyrosine kinase inhibitor. In another six neurons tested, treatment with lavendustin A (10 μM for 2 min after assuming the whole-cell configuration) attenuated the Ang II-induced facilitation of ICa (34.9 ± 3.3% for Ang II only, 13.2 ± 3.2% for Ang II in neurons treated with lavendustin A, n = 20 and 6, respectively).
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A, typical superimposed ICa traces at the times indicated in the time course graph B. B, typical time course of Ang II-induced facilitation of ICa in a neuron treated with genistein (10 μM for 2 min). Genistein (10 μM) and Ang II (1 μM) were bath-applied during the time indicated by the open and filled bars, respectively. C, typical superimposed ICa traces at the times indicated in the time course graph D. D, typical time course of Ang II-induced facilitation of ICa in a neuron treated with PP2 (10 μM for 2 min). PP2 (10 μM) and Ang II (1 μM) were bath-applied during the time indicated by the open and filled bars, respectively. E, typical superimposed ICa traces at the times indicated in the time course graph F. F, typical time course of Ang II-induced facilitation of ICa in a neuron treated with PD98,059 (10 μM for 2 min). PD98,059 (10 μM) and Ang II (1 μM) were bath-applied during the time indicated by the open and filled bars, respectively.
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To evaluate the possible contribution of Src tyrosine kinases to the Ang II-induced facilitation of ICa, the effects of Ang II on ICa in neurons treated with PP2 (a Src tyrosine kinase inhibitor) were investigated. In seven neurons tested, treatment with PP2 (10 μM for 2 min after assuming the whole-cell configuration) attenuated the Ang II-induced facilitation of ICa (34.9 ± 3.3% for Ang II only, 11.2 ± 3.3% for Ang II in neurons treated with PP2, n = 20 and 7, respectively, Figs 5C and D, and 8).
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To evaluate the possible contribution of mitogen-activated protein kinase (MAPK) tyrosine kinases to the Ang II-induced facilitation of ICa, the effects of Ang II on ICa in neurons treated with PD98,059 (a MAPK tyrosine kinase inhibitor) were investigated. In another seven neurons tested, treatment with PD98,059 (10 μM for 2 min after assuming the whole-cell configuration) attenuated the Ang II-induced facilitation of ICa (34.9 ± 3.3% for Ang II only, 14.4 ± 3.1% for Ang II in neurons treated with PD98,059, n = 20 and 7, respectively, Figs 5E and F, and 8).
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It has been reported that genistein acts as a VDCC blocker (Belevych et al. 2002). In fact, genistein blocked ICa from –620 ± 182.5 pA to –501 ± 154 pA. Before application of genistein, the mean percentages of ICa-N, ICa-P/Q, ICa-L and resistance VDCC component (ICa-R) of total ICa were 26.2, 19.4, 39.8 and 15.9%, respectively (n = 4). In the presence of genistein (10 μM for 2 min in bath solution), mean percentages of ICa-N, ICa-P/Q, ICa-L and ICa-R of total ICa were 25.8, 18.5, 41.2 and 16.9%, respectively (n = 5). These results suggest that the abrogated Ang II effect cannot be explained by selective inhibition of ICa-L. Therefore, the effects of Ang II on ICa in neurons dialysed with a pipette solution containing genistein and lavendustin A were investigated. In each of six neurons tested, intracellular application of genistein (50 μM for 7 min after assuming the whole-cell configuration) and lavendustin A (50 μM for 7 min after assuming the whole-cell configuration) attenuated the Ang II-induced facilitation of ICa (31.0 ± 3.2% for Ang II only, 8.4 ± 2.7% for Ang II in neurons dialysed with genistein, 9.2 ± 3.2% for Ang II in neurons dialysed with lavendustin A, n = 5, 6 and 6, respectively, Figs 6A–D, and 8).
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A, typical superimposed ICa traces at the times indicated in the time course graph B. B, typical time course of Ang II-induced facilitation of ICa in a neuron dialysed with a pipette solution containing 50 μM genistein. C, typical superimposed ICa traces at the times indicated in the time course graph D. D, typical time course of Ang II-induced facilitation of ICa in a neuron dialysed with a pipette solution containing 50 μM lavendustin A. Ang II (1 μM) was bath-applied during the time indicated by the filled bar. E, typical superimposed ICa traces at the times indicated in the time course graph F. F, typical time course of Ang II-induced facilitation of ICa in a neuron dialysed with a pipette solution containing 50 μM PD98,059. Ang II (1 μM) was bath-applied during the time indicated by the filled bar.
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The effects of Ang II on ICa in neurons dialysed with a pipette solution containing PD98,059 were investigated. In seven neurons tested, intracellular application of PD98,059 (50 μM for 7 min after assuming the whole-cell configuration) attenuated the Ang II-induced facilitation of ICa (31.0 ± 3.2% for Ang II only, 7.3 ± 3.1% for Ang II in neurons dialysed with PD98,059, n = 5 and 7, respectively, Figs 6E and F, and 8).
These results indicate that Ang II facilitates VDCCs involving Src tyrosine kinase and MAPK in NTS.
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Characterization of MAPK subtypes in Ang II-induced facilitation of ICa
There are three major MAPK subgroups identified, including the extracellular signal-regulated kinases (ERK1/2), p38 MAPK and c-jun-NH2-terminal kinases (JNKs).
To evaluate the possible contribution of ERK1/2 to the Ang II-induced facilitation of ICa, the effects of Ang II on ICa in neurons treated with U0126 (ERK1/2 inhibitor) were investigated. U0126 is a specific inhibitor of MEK1/2 that can block the phosphorylation of p42/p44 MAPK induced by various stimuli (Favata et al. 1998). In six neurons tested, treatment with U0126 (10 μM for 10 min before patch clamp experiments) did not attenuate the Ang II-induced facilitation of ICa (34.9 ± 3.3% for Ang II only, 25.8 ± 2.9% for Ang II in neurons treated with U0126, n = 20 and 6, respectively, Figs 7A and B, and 8).
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A, typical superimposed ICa traces at the times indicated in the time course graph B. B, typical time course of Ang II-induced facilitation of ICa in a neuron treated with U0126 (10 μM for 10 min before patch clamp experiments). C, typical superimposed ICa traces at the times indicated in the time course graph D. D, typical time course of Ang II-induced facilitation of ICa in a neuron treated with SB202190 (10 μM for 10 min before patch clamp experiments). Ang II (1 μM) was bath-applied during the time indicated by the filled bar. E, typical superimposed ICa traces at the times indicated in the time course graph F. F, typical time course of Ang II-induced facilitation of ICa in a neuron treated with SP600125 (10 μM for 10 min). Ang II (1 μM) was bath-applied during the time indicated by the filled bar.
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To evaluate the possible contribution of p38 to the Ang II-induced facilitation of ICa, the effects of Ang II on ICa in neurons treated with SB202190 (p38 MAPK inhibitor) were investigated. The imidazole compound SB202190 is a specific inhibitor of p38 MAPK (Jiang et al. 1996). In seven neurons tested, treatment with SB202190 (10 μM for 10 min before patch clamp experiments) attenuated the Ang II-induced facilitation of ICa (34.9 ± 3.3% for Ang II only, 2.8 ± 1.2% for Ang II in neurons treated with SB202190, n = 20 and 7, respectively, Figs 7C and D, and 8).
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To evaluate the possible contribution of JNK to the Ang II-induced facilitation of ICa, the effects of Ang II on ICa in neurons treated with SP600125 (JNK inhibitor) were investigated. In six neurons tested, treatment with SP600125 (10 μM for 10 min before patch clamp experiments) did not attenuate the Ang II-induced facilitation of ICa (34.9 ± 3.3% for Ang II only, 25.4 ± 2.6% for Ang II in neurons treated with SP600125, n = 20 and 6, respectively, Figs 7E and F, and 8).
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These results indicate that Ang II facilitates VDCCs involving p38 MAPK in NTS.
Discussion
The present study investigated the effects of Ang II on ICa in NTS. While Wang et al. have demonstrated that NADPH oxidase-derived reactive oxygen species (ROS) are involved in Ang II-induced facilitation of L-type VDCCs in NTS (Wang et al. 2004), this is the first demonstration that Ang II facilitates L type VDCCs via Gi-proteins involving Src tyrosine kinase and p38 MAPK kinase mediated by AT1 receptors in NTS. It can be considered that Ang II stimulates Src tyrosine kinase, which in turn activates the p38 MAPK pathways, resulting in facilitation of VDCCs in NTS.
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Ang II modulation of NTS neurons
It is generally accepted that Ang II elicits neuronal excitation of NTS. Several electrophysiological recordings from NTS both in vivo (Hegarty et al. 1996) and in vitro (Barnes et al. 1988, 1991; Qu et al. 1996) described an excitatory effect of Ang II on NTS. Ang II-induced facilitation of VDCCs demonstrated in this study may lead to depolarization of NTS. On the other hand, we cannot rule out the fact that NTS expresses Ca2+-activated K+ (KCa) channels (Tell & Bradley, 1994), which can hyperpolarize the membrane (Halliwell, 1990; Hille, 2001). In fact, several studies in different cells show that Ang II first induces membrane hyperpolarization, before membrane depolarization (Quinn et al. 1987; Chorvatova et al. 1995). This stems from the fact that activation of the AT1 receptor induces facilitation of VDCCs which consequently increases permeability to K+ ions drawing the membrane potential to negative values. Increases in [Ca2+]i induced by Ang II precede this effect and lead to an initial hyperpolarization of the membrane. Afterwards, membrane depolarization occurs until a plateau is reached; then neurons repolarize towards control values. Furthermore, Ang II may very likely not exclusively facilitate VDCCs but also other G-protein-dependent channels. Ang II-induced activation of G-protein-gated inwardly rectifying K+ (GIRK) channels could also be candidates for hyperpolarization. In fact, it has been demonstrated that Ang II modulates several K+ channels (Pan et al. 2001), including GIRK channels (Hoyer et al. 1991) in other tissues. Thus, it will be important to clarify the mechanisms of integration of such G-protein signallings in the neuronal excitation.
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Wang et al. (2002) have demonstrated that application of 2 μM Ang II facilitates L-type VDCCs in NTS. Treatment with MnTBAP (the cell-permeant ROS scavenger) attenuated Ang II -induced facilitation of L-type VDCCs. In addition, treatment with apocynin (an inhibitor of NADPH oxidase assembly) and intracellular dialysis of gp91ds (NADPH oxidase peptide inhibitor) also attenuated Ang II-induced facilitation of L-type VDCCs. These results suggested that NADPH oxidase-derived ROS are involved in Ang II-induced facilitation of L-type VDCCs in NTS. These authors also suggested that such facilitation of L-type VDCCs provides an explanation for the finding that Ang II increases neuronal excitability and spontaneous activity (Ferguson et al. 2001).
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The effect of Ang II on NTS function
There are local renin–angiotensin systems (RASs) in regions such as the kidney and heart, and that dysfunction of these systems may lead to changes in the regulation of blood pressure. Recently, it has been thought likely that all components of the RAS are present in the brain, and local production of angiotensin peptides has been shown in several brain areas (Johnston et al. 1992; Bader & Ganten, 2002). Stimulation of brain RAS leads to increases in blood pressure, attenuation of the baroreceptor heart-rate reflex, stimulation of drinking, and the release of various hormones including vasopressin (Steckelings et al. 1992).
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Several demonstrations suggest that the NTS contains all of the components of RAS including angiotensinogen, angiotensin-converting enzyme (ACE), Ang II and Ang II receptors (Allen et al. 1998). The homeostatic functions of Ang II are mediated, in part, through AT1 receptors in the NTS, which are components of the sensory vagal complex that responds to baroreceptor and volume receptor information by ascending projections to brain regions (Fitzsimons, 1998).
Some reports demonstrated that NTS contributes to baroreceptor regulation. However, the effect of Ang II on the NTS to modulate baroreceptor regulation of blood pressure is complex. Microinjection of Ang II into the NTS has been reported to yield not only pressor, but also depressor effects (Rettig et al. 1986; Mosqueda-Garcia et al. 1990; Fow et al. 1994). Microinjection of low doses of Ang II (200 fmol) into the NTS produced hypotension and bradycardia that mimicked stimulation of the baroreceptor reflex (Fow et al. 1994). Furthermore, high doses of Ang II (1 pmol) microinjected into the NTS increased arterial pressure (Casto & Phillips, 1984; Rettig et al. 1986). The exact mechanisms by which Ang II produces pressor or depressor responses in the NTS are unknown. The activation of different receptor subtypes and affinities may be due to opposite effects of this peptide (Andresen, 1994; Fow et al. 1994). Alternatively, it can be considered that neuronal networks exist within the NTS. NTS neurons can be divided into two groups, GABAergic and glutamatergic (Mifflin & Felder, 1990; Brooks et al. 1992). As mentioned above, microinjection of Ang II into NTS reduces baroreflex function. It can be considered that facilitation of VDCCs on GABAergic neurons can enhance GABA release and therefore a reduced baroreflex function. Wong et al. (2002) proposed an enhancement of GABA release induced by NO that was generated by Ca2+ activation of NOS. The increased Ca2+ influx through L-type VDCCs could activate the same mechanism. However, in the case of glutamatergic neurons, it would have the opposite effect. In this study, it is not possible to identify distinct NTS subgroups.
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Ang II-induced facilitation of VDCCs involving Src
Protein tyrosine kinases (PTKs) are classified into two major groups: receptor-linked PTKs (RTKs) and non-receptor-linked PTKs (non-RTKs). RTKs, such as receptors for growth factor (e.g. platelet-derived growth factor (PDGF), or epidermal growth factor (EGF)), are attached to the receptor as a special domain. Interaction of a specific ligand with the receptor results in dimerization of the receptor, auto-phosphorylation of specific tyrosine residues, and expression of tyrosine kinase activity for specific substrates. In the other class, the non-RTKs such as Pyk, FAK, JAK, Src and products of Abl and Fes are cytoplasmic enzymes.
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Ang II-induced facilitation of VDCCs suggests that a principal effect of Ang II modulation might be regulation of Ca2+-dependent enzymes, in particular, transcription factors (Alberini et al. 1994). L-type VDCCs have been linked to not only somal action potentials, but also to the activation of Src, the modulation of RTK (Finkbeiner & Greenberg, 1996), and gene expression, including synthesis of ion channels (Murphy et al. 1991; Misra et al. 1994). During differentiation, L-type VDCC-mediated Ca2+ influx is essential for neurite outgrowth, synapse formation, survival and the shift to the mature action potential profile (Spitzer, 1994). Developmentally, Ca2+ influx would be expected to trigger influx-dependent cellular differentiation (Spitzer, 1994; Gu & Spitzer, 1995) and, because early action potential activity has been linked to establishing neuronal circuits (Katz & Shatz, 1996), potentially could contribute to pattern information. These studies strongly imply that Ang II-induced facilitation of L-type VDCCs would lead to a substantial increase in Ca2+ influx. Whether occurring as a result of spontaneous activity or mature action potential firing, such increases in [Ca2+]i may dramatically alter neuronal function.
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As shown in Fig. 5B, D and F, even under basal conditions, ICa could be attenuated by tyrosine kinase inhibitors. Precisely how Src kinase modulates the VDCCs is uncertain. Single channel studies in inside-out patches isolated from rat portal vein cells have demonstrated that genistein reduces the open probability of L-type VDCCs without altering unit amplitude or slope conductance (Liu & Sperelakis, 1997). A similar effect of genistein on steady-state inactivation of ICa has also been reported in other cell types (Yokoshiki et al. 1995; Liu et al. 1997). In addition, Fitzgerald & Dolphin (1997) have suggested that Src kinase can influence the gating of VDCCs. These data could be taken to implicate Src tyrosine kinase and/or MAPK kinase in modulating the inactivation process of VDCCs and imply a possible role for tyrosine phosphorylation/dephosphorylation in VDCC inactivation. Therefore, it can be considered that this kinase may represent a ubiquitous modulator of ion channels in many cell types.
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There is evidence for c-src being responsible for the modulation of L-type VDCCs in vascular smooth muscle, particularly when taken in conjunction with previous observations which have shown that VDCCs are facilitated by intracellular application of purified c-src (Wijetunge & Hughes, 1995), or a peptide that activates endogenous c-src (Wijetunge & Hughes, 1996). The idea that c-src may regulate L-type VDCCs receives further support from the observation that c-src can be found in immunoprecipitates of the 1c subunit of L-type VDCCs (Hu et al. 1998). Hence there is both functional and biochemical evidence closely linking c-src to the pore-forming 1c subunit of L-type VDCCs.
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Furthermore, L-type VDCCs have been implicated in the synaptic control of nuclear gene expression. Ang II has been shown to increase the expression of inducible transcription factors mediated by AT1 receptor-dependent mechanisms in the brain regions (Moellenhoff et al. 1998). The possibility exists that VDCCs play a role in regulating gene expression in these situations, thus serving as a focal point for Ang II-induced regulation of both neuronal excitability and protein expression. The idea that Src tyrosine kinase may regulate L-type VDCCs receives further support from the observation that Src tyrosine kinase can be found in immunoprecipitates of the 1c subunit of L-type VDCCs (Hu et al. 1998).
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In this study the EC50 was 167 nM, and the Hill slope coefficient was 1.7 in NTS. EC50 values for Ang II-induced facilitation of ICa have been calculated in other tissues. In rabbit ventricular myocytes (0.75 nM, Ichiyanagi et al. 2002) and rat heart myocytes (0.6 nM, Allen et al. 1988) it was found to be 100-fold more potent than in NTS. The discrepancy observed in these studies carried out in different cell types may be due to cell-dependent differences in the regulation of membrane excitability.
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As shown in Fig. 8, genistein and PP2 failed to completely block the response of Ang II. What other kinases/mechanisms are involved in the regulation of the opening of VDCCsIt has been reported that arachidonic acid (AA) is a second messenger in the regulation of VDCCs. Several GPCRs can activate phospholipase A (PLA), which in turn catalyses the release of AA (Axelrod et al. 1988). AA-induced facilitation of VDCCs has been reported (Vacher et al. 1989; Huang et al. 1992; Chesnoy-Marchais & Fritsch, 1994). Ang II-induced AA release has been demonstrated recently (Li & Malik, 2005). Calcium–calmodulin protein kinase II (CaMKII) may also have a role in the AA-induced facilitation of VDCCs: it has been reported that Ang II-induced inhibition of Kv channel current involved CaMKII (Sun et al. 2003). Therefore, the Ang II receptor pathway should be investigated in a further study.
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