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Protein kinase C activation-induced increases of neural activity are enhanced in the hypothalamus of spontaneously hypertensive rats
Brain Research 1033 (2005) 157 – 163 www.elsevier.com/locate/brainres
Research report
Protein kinase C activation-induced increases of neural activity are enhanced in the hypothalamus of spontaneously hypertensive rats Takao Kubo*, Yukihiko Hagiwara Department of Pharmacology, Showa Pharmaceutical University, Higasi-tamagawagakuen, Machida, Tokyo 194-8543, Japan Accepted 19 November 2004 Available online 11 January 2005
Abstract We have previously reported that some neurons in the anterior hypothalamic area (AHA) are tonically activated by endogenous angiotensins in rats and that activities of these angiotensin II-sensitive neurons in the AHA are enhanced in spontaneously hypertensive rats (SHR). In addition, neural activations induced by both angiotensin II and glutamate were enhanced in the AHA of SHR. In this study, we examined whether intracellular neural activation mechanisms via protein kinase C (PKC) and a potassium channel are altered in angiotensin II-sensitive neurons in the AHA of SHR. Male 15- to 16-week-old SHR and age-matched Wistar–Kyoto rats (WKY) and Wistar rats were anesthetized and artificially ventilated. Extracellular potentials were recorded from single neurons in the AHA. Pressure application of the PKC activator phorbol 12-myristate 13-acetate (PMA) onto angiotensin II-sensitive neurons in the AHA of Wistar rats increased their firing rate. The increase of unit activity by PMA was inhibited by the potent inhibitor of PKC, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), but not by the weak PKC inhibitor, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride (HA1004). The increase of unit firing by PMA was enhanced in SHR as compared with WKY. Pressure application of H-7 alone decreased the basal firing activity of angiotensin II-sensitive neurons in SHR but not in WKY. HA1004 did not affect the basal firing activity of angiotensin II-sensitive neurons in SHR. Angiotensin II-induced increases of firing rate in AHA neurons were inhibited by H-7 and the inhibition by H-7 was enhanced in SHR as compared with WKY. Pressure application of 4-aminopyridine, a blocker of the transient potassium current, onto angiotensin II-sensitive neurons increased their firing rate and the increase of unit firing rate was almost the same in WKY and SHR. These findings indicate that activation of PKC increases neural activity in angiotensin II-sensitive neurons in the AHA and that this PKC activationinduced increase of neural activity is enhanced in the AHA of SHR. It seems likely that the enhanced PKC activation effect is responsible for the enhanced basal neural activity seen in the AHA of SHR. D 2004 Elsevier B.V. All rights reserved. Theme: Endocrine and autonomic regulation Topic: Cardiovascular regulation Keywords: Angiotensinergic; Protein kinase C; Hypothalamus; Unit discharge; Spontaneously hypertensive rats
1. Introduction Central angiotensin II, especially hypothalamic angiotensin II, has been implicated in the development of hypertension in spontaneously hypertensive rats (SHR), a genetic model for hypertension [3,4,7,11,14,16–18,24]. Microinjec-
* Corresponding author. Fax: +81 42 721 1588. E-mail address: [email protected] (T. Kubo). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.11.029
tion of angiotensin II into the anterior hypothalamic area (AHA) causes enhanced pressor responses in conscious SHR as compared with Wistar–Kyoto rats (WKY) and injection of the angiotensin type 1 (AT1) receptor antagonist losartan into the AHA causes depressor responses in SHR but not in WKY [14,15], suggesting that an angiotensin system in the AHA is enhanced in SHR and that this enhancement is involved in hypertension in this strain. Recently, we have demonstrated that pressure-ejected application of angiotensin II and glutamate onto some
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neurons in the AHA of rats increases firing rate [5]. The increase of unit firing induced by angiotensin II but not by glutamate was inhibited by losartan applied onto the same neurons. Application of losartan alone decreased the basal firing rate of angiotensin II-sensitive neurons in the AHA. These findings suggest that some neurons in the AHA are tonically activated by endogenous angiotensins. In SHR, the basal firing rate of angiotensin II-sensitive neurons was increased as compared with WKY [12]. The increase of unit firing by angiotensin II was also enhanced in SHR as compared with WKY. These findings suggest that activities of angiotensin II-sensitive neurons in the AHA are enhanced in SHR and that this enhanced activity is, at least in part, due to enhanced neural reactivity to angiotensin II. In addition, we have found that the increase of unit firing of angiotensin II-sensitive neurons induced by glutamate is also enhanced in SHR as compared with WKY [12]. One possible explanation for these enhanced reactivities to both angiotensin II and glutamate is that intracellular signal transduction mechanisms are enhanced in AHA neurons of SHR. For example, activation of either AT1 receptors or glutamate receptors in neurons stimulates phosphoinositide hydrolysis, resulting in increased PKC activity [1,6,20,22,25]. In neurons, PKC activation causes both the reduction in the delayed rectifier K+ current (I k) and in the transient potassium current (IA), and stimulates calcium current (I Ca) [20,22], resulting in an increase of neural activity. In the present study, we examined whether intracellular neural activation mechanisms via PKC are altered in angiotensin II-sensitive neurons in the AHA of SHR. For comparison, we also examined whether intracellular neuronal activation via blockade of the transient potassium current is altered in angiotensin II-sensitive neurons in the AHA of SHR.
2. Materials and methods Studies were conducted using 15- to 16-week-old male SHR and age-matched WKY Izumo strain rats (SHR/Izm and WKY/Izm, respectively) maintained by the Disease Model Cooperative Research Association, Kyoto, Japan. The tail cuff measurements revealed that the SHR had a significantly higher systolic blood pressure (183 F 1 mm Hg) than did WKY (130 F 1 mm Hg)( P b 0.05) under conscious condition. In some experiments, Wistar rats (300–360 g) were used. They were kept in cages in a room with a 12-h light–dark cycle. Animals were fed standard laboratory rat chow and tap water ad libitum. All procedures were done in accordance with the guidelines outlined by the Institutional Animal Care and Use Committee of the Showa. All efforts were made to minimize animal suffering. Animals were given pentobarbital, 50 mg/kg, intraperitoneally, and 15 mg/kg was injected subcutaneously
every 1 h from 60 min after the first injection. The femoral artery and vein were cannulated. The rats were placed in a stereotaxic apparatus and ventilated artificially with a respirator. Tidal volumes were chosen according to the ventilation standards for small mammals [10] and end-tidal pCO2 levels were monitored using a clinical gas monitor (San-ei, 1H26). The end-tidal pCO2 and rectal temperature were kept within 3.5–4.5% and 36–378C, respectively. The basal mean arterial pressure was 90 F 2 mm Hg (n = 41) and 122 F 2 mm Hg (n = 54) in pentobarbital-anesthetized WKY and SHR, respectively ( P b 0.05). Extracellular single-unit spontaneous activity of neurons was recorded from the AHA (1.3 mm caudal and 0.8 mm lateral to the bregma, and 8.2 mm below the cerebral surface), as described [14]. Extracellular recording was performed through the electrode, which was connected to a preamplifier (Model 12317, Nihondenki San-ei Instrument Co., Ltd), and the spike potentials of the neurons were measured by means of a window discriminator. Electrical activity was displayed on a medical oscilloscope with an audiometer and filtered (band pass 0.1–10 kHz). A signal processor (Model 7T08, Nihondenki San-ei Instrument Co., Ltd.) was used for compiling the data in the form of integrated rate histograms. Pressure-ejection experiments utilized three-barrel glass microelectrodes both to record the extracellular potentials from single neurons and to apply drugs at the recording site as described [13]. Drugs were pressure-ejected from micropipettes by applying compressed nitrogen gas, which was regulated 10 psi at a pneumatic valve, to the electrode assembly via high pressure (Neuro Phore BH-2 System, Medical Systems Corp. Ltd., NY). The basal unit firing rate of neurons was obtained by averaging firing rates for 1 min. The increase of drug-induced firing rate was obtained by averaging drug-induced increases of firing rate for 5 s. We made the drug application 2–3 times using one electrode, but there was no tachyphylaxis. The site of unit recording was stained by expelling the pontamine sky blue from the electrode by the passage of 20–50 AA current for about 15 min. The brain was removed, frozen sections were cut (50 Am), and the recording sites were identified. Drugs used were angiotensin II acetate salt, phorbol 12myristate 13-acetate (PMA), 1-(5-isoquinolinesulfonyl)-2methylpiperazine dihydrochloride (H-7), N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride (HA1004), and 4-aminopyridine (Sigma, St. Louis, MO, USA). All drugs except PMA were dissolved in artificial cerebrospinal fluid (in mmol/L): NaCl, 119; KCl, 3.3; CaCl2, 1.3; MgCl2, 1.2; Na2HPO4, 0.5; NaHCO3, 21.0; glucose, 3.4 (pH 7.4). PMA was dissolved in DMSO to an initial concentration of 1 mM and then diluted in the artificial cerebral spinal fluid to the appropriate concentrations. The results are expressed as mean F SEM. All results were analyzed by either Student’s t test or one-way analysis of variance combined with Dunnett’s test for post
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neurons if pressure-ejected application (10 psi) of angiotensin II (10 7 M) onto neurons increased the firing rate of the neurons. Pressure-ejected application (10 psi for 20 s) of the PKC activator phorbol 12-myristate 13-acetate (PMA)(10 6 M) onto angiotensin II-sensitive neurons increased the firing rate (Figs. 1A, B, and C). Onset was around 30 s after the beginning of PMA application (Fig. 1A). Solvent for PMA (10 6 M) did not affect the firing rate in all AHA neurons tested (n = 7 in 7 rats) (data not shown). Pressure application of the PKC inhibitor H-7 (10 5 and 10 4 M) onto AHA neurons inhibited the increase of unit firing induced by PMA (10 6 M) in a concentrationdependent manner (Figs. 1B and D). On the other hand, application of HA1004 (10 4 M), a negative control of H-7, onto AHA neurons did not affect the increase of unit firing induced by PMA (10 6 M) (Figs. 1C and D). 3.2. Effects of PMA, H-7, and HA1004 on angiotensin II-sensitive neurons in the AHA of WKY and SHR The basal firing rate of angiotensin II-sensitive neurons was 7.1 F 0.1 spikes/s (n = 85 in 54 rats) in SHR as compared to 4.2 F 0.1 spikes/s (n = 64 in 41 rats) in WKY ( P b 0.05). Pressure application (10 psi for 20 s) of PMA (10 7 and 10 6 M) increased the firing rate of angiotensin II-sensitive neurons in the AHA of WKY and SHR (Fig. 2). The increase of firing rate by PMA was greater in SHR than in
Fig. 1. (A) Firing of an angiotensin II-sensitive neurons before, during, and after pressure application (10 psi for 20 s) of PMA (10 6 M) in the AHA of a Wistar rat. Application of PMA is indicated by underlines. Figures under unit firing show the time (s) before and after the beginning of PMA application. Calibration: the vertical bar represents 1 mV and the horizontal bar represents 1 s. (B and C) Effects of pressure application (10 psi) of H-7 (B) and HA1004 (C) on the PMA (10 psi for 20 s)-induced firing responses of neurons in the AHA of Wistar rats. (D) Effects of pressure application (10 psi) of H-7 and HA1004 on PMA-induced increases of neural firing rate in the AHA of Wistar rats. Treatment with H-7 or HA1004 was started around 1 min before pressure application (10 psi for 20 s) of PMA. Open columns, before treatment; dotted columns, after treatment. Values are the mean F SEM of 7–8 neurons from five rats. *P b 0.05, compared to before treatment.
hoc analysis for intergroup comparison. Differences were considered significant at P b 0.05.
3. Results 3.1. Effects of PMA, H-7, and HA1004 on neurons in the AHA of Wistar rats The basal mean arterial pressure was 91 F 2 mm Hg (n = 30) in pentobarbital-anesthetized rats. Individual neurons in the AHA were determined to be angiotensin II-sensitive
Fig. 2. (A–D) Firing responses of angiotensin II-sensitive neurons to pressure application (10 psi for 20 s) of PMA in the AHA of WKY (A and B) and SHR (C and D). (E) Effects of pressure application (10 psi for 20 s) of PMA on the firing rate of angiotensin II-sensitive neurons in the AHA of WKY and SHR. Open columns, before treatment; dotted columns, after treatment. Values are the mean F SEM of 12–14 neurons from eight rats. *P b 0.05, compared to WKY.
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WKY. Application (10 psi for 60 s) of H-7 (10 4 M) onto angiotensin II-sensitive neurons decreased the amplitude of unit firing by 65.8 F 3.5% in all neurons tested in the AHA of SHR (12 neurons from 7 rats), whereas the inhibitor (10 4 M) did not affect it in all neurons tested in the AHA of WKY (10 neurons from 7 rats) (Figs. 3A and B). The decrease in amplitude of firing by H-7 in SHR began around 1 min and lasted for about 3 min after the beginning of H-7 application. The firing rate of angiotensin II-sensitive neurons was not changed by H-7 in both SHR and WKY (Figs. 3C and D). Application (10 psi for 60 s) of HA1004 (10 4 M), a negative control of H-7, onto angiotensin IIsensitive neurons did not affect the firing rate in all neurons tested in the AHA of SHR (10 neurons from 7 rats) (data not shown). Pressure application (10 psi for 5 s) of angiotensin II (10 7 M) onto AHA neurons increased the firing rate in WKY and SHR. In SHR, application of H-7 (10 5 and 10 4 M) onto angiotensin II-sensitive neurons inhibited the increase of firing rate induced by angiotensin II (10 7 M)
in a concentration-dependent manner (Fig. 4A). On the other hand, in WKY, H-7 (10 5 M) did not affect the angiotensin II-induced increase of firing rate, although H-7 (10 4 M) significantly inhibited the increase of firing rate by angiotensin II. Pressure application (10 psi) of vehicle (5–60 s) consistently did not affect the basal amplitude and firing rate (n =21, data not shown), suggesting that the effects found by pressure application of drugs are not due to distortion. Fig. 4B shows the location of recording sites for angiotensin II-sensitive neurons in the AHA of WKY and SHR. Recording sites were located in an area of the AHA. The recording sites for the SHR were similar to those for WKY. 3.3. Effects of 4-aminopyridine on angiotensin II-sensitive neurons in the AHA of WKY and SHR Pressure application (10 psi for 5 s) of 4-aminopyridine (10 4 and 310 4 M), a blocking agent of the transient
Fig. 3. Firing (A and B) and continuous rate-meter recordings (C and D) of angiotensin II-sensitive neurons before, during, and after pressure application (10 psi for 60 s) of H-7 (10 4 M) in the AHA of WKY (A and C) and SHR (B and D). In A and B, applications of H-7 are indicated by underlines. Calibrations in A and B: vertical bars represent 1 mV and horizontal bars represent 1 s.
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angiotensin II-sensitive neurons in the AHA of SHR, whereas this agent did not affect neural activity in the AHA of WKY. In addition, HA1004 did not affect neural activity in the AHA of SHR. These findings are compatible with the idea that intracellular signal transduction mechanisms via PKC are enhanced in angiotensin II-sensitive neurons in the AHA of SHR and that the enhanced intracellular signal transduction mechanisms via PKC are involved in the basal-enhanced neural activity of AHA angiotensin II-sensitive neurons seen in SHR [12]. In a previous study, we demonstrated that tonically released endogenous angiotensins are involved in basal neural activity of angiotensin II-sensitive neurons in the AHA [5]. Thus, it could be expected that exogenously applied angiotensin II-induced activation of AHA neurons is also inhibited by H-7 and that inhibition of neural activation by H-7 is greater in SHR than in WKY. In the present study, indeed, we found that H-7 (10 5 M) inhibited angiotensin II-induced neural activation in SHR but not in WKY. In addition, H-7 (10 4 M) caused a greater inhibition of angiotensin II-induced neural activation in SHR than in WKY, whereas HA1004 (10 4 M) did not affect the angiotensin II-induced neural activation in SHR. In the previous study, both angiotensin II and glutamate caused greater neural activation in the AHA of SHR [12]. Fig. 4. (A) Effects of pressure application (10 psi) of H-7 or HA1004 on the angiotensin II (10 7 M, 10 psi for 5 s)-induced increase of neural firing rate in the AHA of WKY and SHR. Treatment with H-7 or HA1004 was started around 1 min before application of angiotensin II. Open columns, before treatment; dotted columns, after treatment. Values are the mean F SEM of 8–10 neurons from six rats. *P b 0.05, compared to WKY. (B) Diagrams illustrating the location of recording sites for angiotensin II-sensitive neurons in the AHA of WKY and SHR.
potassium current, onto angiotensin II-sensitive neurons increased the firing rate in the AHA of WKY and SHR (Fig. 5). The increases of firing rate by 4-aminopyridine were almost the same in WKY and SHR.
4. Discussion In the present study, the PKC activator PMA caused an increase of neural activity in the AHA of Wistar rats, and the PMA-induced neural activation was inhibited by H-7 but not by HA1004. The inhibitory effect of H-7 on PKC is greater than that of HA 1004, while the inhibitory effects of H-7 on cGMP-dependent protein kinase and cAMP-dependent protein kinase are rather smaller than those of HA 1004 [2,8]. Thus, the results of the present study suggest the involvement of PKC in the PMAinduced neural activation. In the present study, the PMA-induced neural activation of AHA angiotensin II-sensitive neurons was greater in SHR than in WKY. H-7 alone inhibited neural activity of
Fig. 5. (A–D) Firing responses of angiotensin II-sensitive neurons to pressure application (10 psi for 5 s) of 4-aminopyridine (4AP) in the AHA of WKY (A and B) and SHR (C and D). (E) Effects of pressure application (10 psi for 5 s) of 4-aminopyridine (4AP) on the firing rate of angiotensin II-sensitive neurons in the AHA of WKY and SHR. Open columns, WKY; dotted columns, SHR. Values are the mean F SEM of 9–10 neurons from six rats.
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Both agonists increase PKC activity, resulting in an increase of neural activity [20]. Thus, the enhanced intracellular signal transduction mechanisms via PKC in AHA neurons of SHR found in this study could, at least in part, explain the enhanced neural reactivity to both angiotensin II and glutamate. PKC activation causes both the reduction in the delayed rectifier K+ current (I k) and in the transient potassium current (I A), and stimulates calcium current (I Ca), resulting in an increase of neural activity [20,22]. In the present study, blockade of I A by 4-aminopyridine increased the unit firing rate of angiotensin II-sensitive neurons in the AHA of WKY and SHR, and the increase of firing rate by 4-aminopyridine was almost the same in the AHA of both kinds of rats. Thus, we could not find any alteration in intracellular mechanisms via I A in AHA neurons of SHR. In the present study, H-7 inhibited both basal neural activity and angiotensin II-induced neural activation in the AHA of SHR, suggesting that PKC is involved in neural activation caused by both spontaneously released and exogenously applied angiotensins in SHR. In contrast, in the AHA of WKY, H-7 did not affect basal neural activity, whereas it caused a slight but significant inhibition of angiotensin II-induced increase of neural activity. This discrepancy could be explained by assuming that in WKY, PKC is not involved in neural activation caused by a small amount of angiotensins like spontaneously released angiotensins, but this enzyme is involved in neural activation caused by a large amount of angiotensin II like exogenously applied angiotensin II (10 7 M). Clearly, more studies will be needed to clarify why H-7 does not affect basal neural activity in the AHA of WKY. It has been reported that total PKC activity is significantly increased in synaptosomal samples isolated from the forebrain, midbrain, and hind brain of SHR [9]. Tsuda et al. [21] have demonstrated that H-7 reduces the stimulationevoked [3H]-acetylcholine release to a greater extent in superfused slices of striatum from SHR than in those of WKY, suggesting that the PKC system may participate in the central nervous system in hypertension. These findings are compatible with the results of the present study. On the other hand, many papers have demonstrated various alterations other than a defect in PKC in the brain of SHR [19,23]. Thus, it will be next needed to investigate that these alterations found in the brain of SHR are related each other or independent. In summary, this study demonstrates that activation of PKC increases neural activity of angiotensin II-sensitive neurons in the AHA and that this PKC activation-induced increase of neural activity is enhanced in the AHA of SHR. Blockade of PKC activity by H-7 caused a decrease of basal neural activity of angiotensin II-sensitive neurons in the AHA of SHR but not of WKY, suggesting that the enhanced PKC activation effect may be responsible for the enhanced basal neural activity seen in the AHA of SHR.
Acknowledgment This study was supported in part by a Grant-in-Aid for Scientific Research (No. 13672300) from Japan Society for the Promotion of Science.
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Research report
Protein kinase C activation-induced increases of neural activity are enhanced in the hypothalamus of spontaneously hypertensive rats Takao Kubo*, Yukihiko Hagiwara Department of Pharmacology, Showa Pharmaceutical University, Higasi-tamagawagakuen, Machida, Tokyo 194-8543, Japan Accepted 19 November 2004 Available online 11 January 2005
Abstract We have previously reported that some neurons in the anterior hypothalamic area (AHA) are tonically activated by endogenous angiotensins in rats and that activities of these angiotensin II-sensitive neurons in the AHA are enhanced in spontaneously hypertensive rats (SHR). In addition, neural activations induced by both angiotensin II and glutamate were enhanced in the AHA of SHR. In this study, we examined whether intracellular neural activation mechanisms via protein kinase C (PKC) and a potassium channel are altered in angiotensin II-sensitive neurons in the AHA of SHR. Male 15- to 16-week-old SHR and age-matched Wistar–Kyoto rats (WKY) and Wistar rats were anesthetized and artificially ventilated. Extracellular potentials were recorded from single neurons in the AHA. Pressure application of the PKC activator phorbol 12-myristate 13-acetate (PMA) onto angiotensin II-sensitive neurons in the AHA of Wistar rats increased their firing rate. The increase of unit activity by PMA was inhibited by the potent inhibitor of PKC, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), but not by the weak PKC inhibitor, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride (HA1004). The increase of unit firing by PMA was enhanced in SHR as compared with WKY. Pressure application of H-7 alone decreased the basal firing activity of angiotensin II-sensitive neurons in SHR but not in WKY. HA1004 did not affect the basal firing activity of angiotensin II-sensitive neurons in SHR. Angiotensin II-induced increases of firing rate in AHA neurons were inhibited by H-7 and the inhibition by H-7 was enhanced in SHR as compared with WKY. Pressure application of 4-aminopyridine, a blocker of the transient potassium current, onto angiotensin II-sensitive neurons increased their firing rate and the increase of unit firing rate was almost the same in WKY and SHR. These findings indicate that activation of PKC increases neural activity in angiotensin II-sensitive neurons in the AHA and that this PKC activationinduced increase of neural activity is enhanced in the AHA of SHR. It seems likely that the enhanced PKC activation effect is responsible for the enhanced basal neural activity seen in the AHA of SHR. D 2004 Elsevier B.V. All rights reserved. Theme: Endocrine and autonomic regulation Topic: Cardiovascular regulation Keywords: Angiotensinergic; Protein kinase C; Hypothalamus; Unit discharge; Spontaneously hypertensive rats
1. Introduction Central angiotensin II, especially hypothalamic angiotensin II, has been implicated in the development of hypertension in spontaneously hypertensive rats (SHR), a genetic model for hypertension [3,4,7,11,14,16–18,24]. Microinjec-
* Corresponding author. Fax: +81 42 721 1588. E-mail address: [email protected] (T. Kubo). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.11.029
tion of angiotensin II into the anterior hypothalamic area (AHA) causes enhanced pressor responses in conscious SHR as compared with Wistar–Kyoto rats (WKY) and injection of the angiotensin type 1 (AT1) receptor antagonist losartan into the AHA causes depressor responses in SHR but not in WKY [14,15], suggesting that an angiotensin system in the AHA is enhanced in SHR and that this enhancement is involved in hypertension in this strain. Recently, we have demonstrated that pressure-ejected application of angiotensin II and glutamate onto some
158
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neurons in the AHA of rats increases firing rate [5]. The increase of unit firing induced by angiotensin II but not by glutamate was inhibited by losartan applied onto the same neurons. Application of losartan alone decreased the basal firing rate of angiotensin II-sensitive neurons in the AHA. These findings suggest that some neurons in the AHA are tonically activated by endogenous angiotensins. In SHR, the basal firing rate of angiotensin II-sensitive neurons was increased as compared with WKY [12]. The increase of unit firing by angiotensin II was also enhanced in SHR as compared with WKY. These findings suggest that activities of angiotensin II-sensitive neurons in the AHA are enhanced in SHR and that this enhanced activity is, at least in part, due to enhanced neural reactivity to angiotensin II. In addition, we have found that the increase of unit firing of angiotensin II-sensitive neurons induced by glutamate is also enhanced in SHR as compared with WKY [12]. One possible explanation for these enhanced reactivities to both angiotensin II and glutamate is that intracellular signal transduction mechanisms are enhanced in AHA neurons of SHR. For example, activation of either AT1 receptors or glutamate receptors in neurons stimulates phosphoinositide hydrolysis, resulting in increased PKC activity [1,6,20,22,25]. In neurons, PKC activation causes both the reduction in the delayed rectifier K+ current (I k) and in the transient potassium current (IA), and stimulates calcium current (I Ca) [20,22], resulting in an increase of neural activity. In the present study, we examined whether intracellular neural activation mechanisms via PKC are altered in angiotensin II-sensitive neurons in the AHA of SHR. For comparison, we also examined whether intracellular neuronal activation via blockade of the transient potassium current is altered in angiotensin II-sensitive neurons in the AHA of SHR.
2. Materials and methods Studies were conducted using 15- to 16-week-old male SHR and age-matched WKY Izumo strain rats (SHR/Izm and WKY/Izm, respectively) maintained by the Disease Model Cooperative Research Association, Kyoto, Japan. The tail cuff measurements revealed that the SHR had a significantly higher systolic blood pressure (183 F 1 mm Hg) than did WKY (130 F 1 mm Hg)( P b 0.05) under conscious condition. In some experiments, Wistar rats (300–360 g) were used. They were kept in cages in a room with a 12-h light–dark cycle. Animals were fed standard laboratory rat chow and tap water ad libitum. All procedures were done in accordance with the guidelines outlined by the Institutional Animal Care and Use Committee of the Showa. All efforts were made to minimize animal suffering. Animals were given pentobarbital, 50 mg/kg, intraperitoneally, and 15 mg/kg was injected subcutaneously
every 1 h from 60 min after the first injection. The femoral artery and vein were cannulated. The rats were placed in a stereotaxic apparatus and ventilated artificially with a respirator. Tidal volumes were chosen according to the ventilation standards for small mammals [10] and end-tidal pCO2 levels were monitored using a clinical gas monitor (San-ei, 1H26). The end-tidal pCO2 and rectal temperature were kept within 3.5–4.5% and 36–378C, respectively. The basal mean arterial pressure was 90 F 2 mm Hg (n = 41) and 122 F 2 mm Hg (n = 54) in pentobarbital-anesthetized WKY and SHR, respectively ( P b 0.05). Extracellular single-unit spontaneous activity of neurons was recorded from the AHA (1.3 mm caudal and 0.8 mm lateral to the bregma, and 8.2 mm below the cerebral surface), as described [14]. Extracellular recording was performed through the electrode, which was connected to a preamplifier (Model 12317, Nihondenki San-ei Instrument Co., Ltd), and the spike potentials of the neurons were measured by means of a window discriminator. Electrical activity was displayed on a medical oscilloscope with an audiometer and filtered (band pass 0.1–10 kHz). A signal processor (Model 7T08, Nihondenki San-ei Instrument Co., Ltd.) was used for compiling the data in the form of integrated rate histograms. Pressure-ejection experiments utilized three-barrel glass microelectrodes both to record the extracellular potentials from single neurons and to apply drugs at the recording site as described [13]. Drugs were pressure-ejected from micropipettes by applying compressed nitrogen gas, which was regulated 10 psi at a pneumatic valve, to the electrode assembly via high pressure (Neuro Phore BH-2 System, Medical Systems Corp. Ltd., NY). The basal unit firing rate of neurons was obtained by averaging firing rates for 1 min. The increase of drug-induced firing rate was obtained by averaging drug-induced increases of firing rate for 5 s. We made the drug application 2–3 times using one electrode, but there was no tachyphylaxis. The site of unit recording was stained by expelling the pontamine sky blue from the electrode by the passage of 20–50 AA current for about 15 min. The brain was removed, frozen sections were cut (50 Am), and the recording sites were identified. Drugs used were angiotensin II acetate salt, phorbol 12myristate 13-acetate (PMA), 1-(5-isoquinolinesulfonyl)-2methylpiperazine dihydrochloride (H-7), N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride (HA1004), and 4-aminopyridine (Sigma, St. Louis, MO, USA). All drugs except PMA were dissolved in artificial cerebrospinal fluid (in mmol/L): NaCl, 119; KCl, 3.3; CaCl2, 1.3; MgCl2, 1.2; Na2HPO4, 0.5; NaHCO3, 21.0; glucose, 3.4 (pH 7.4). PMA was dissolved in DMSO to an initial concentration of 1 mM and then diluted in the artificial cerebral spinal fluid to the appropriate concentrations. The results are expressed as mean F SEM. All results were analyzed by either Student’s t test or one-way analysis of variance combined with Dunnett’s test for post
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neurons if pressure-ejected application (10 psi) of angiotensin II (10 7 M) onto neurons increased the firing rate of the neurons. Pressure-ejected application (10 psi for 20 s) of the PKC activator phorbol 12-myristate 13-acetate (PMA)(10 6 M) onto angiotensin II-sensitive neurons increased the firing rate (Figs. 1A, B, and C). Onset was around 30 s after the beginning of PMA application (Fig. 1A). Solvent for PMA (10 6 M) did not affect the firing rate in all AHA neurons tested (n = 7 in 7 rats) (data not shown). Pressure application of the PKC inhibitor H-7 (10 5 and 10 4 M) onto AHA neurons inhibited the increase of unit firing induced by PMA (10 6 M) in a concentrationdependent manner (Figs. 1B and D). On the other hand, application of HA1004 (10 4 M), a negative control of H-7, onto AHA neurons did not affect the increase of unit firing induced by PMA (10 6 M) (Figs. 1C and D). 3.2. Effects of PMA, H-7, and HA1004 on angiotensin II-sensitive neurons in the AHA of WKY and SHR The basal firing rate of angiotensin II-sensitive neurons was 7.1 F 0.1 spikes/s (n = 85 in 54 rats) in SHR as compared to 4.2 F 0.1 spikes/s (n = 64 in 41 rats) in WKY ( P b 0.05). Pressure application (10 psi for 20 s) of PMA (10 7 and 10 6 M) increased the firing rate of angiotensin II-sensitive neurons in the AHA of WKY and SHR (Fig. 2). The increase of firing rate by PMA was greater in SHR than in
Fig. 1. (A) Firing of an angiotensin II-sensitive neurons before, during, and after pressure application (10 psi for 20 s) of PMA (10 6 M) in the AHA of a Wistar rat. Application of PMA is indicated by underlines. Figures under unit firing show the time (s) before and after the beginning of PMA application. Calibration: the vertical bar represents 1 mV and the horizontal bar represents 1 s. (B and C) Effects of pressure application (10 psi) of H-7 (B) and HA1004 (C) on the PMA (10 psi for 20 s)-induced firing responses of neurons in the AHA of Wistar rats. (D) Effects of pressure application (10 psi) of H-7 and HA1004 on PMA-induced increases of neural firing rate in the AHA of Wistar rats. Treatment with H-7 or HA1004 was started around 1 min before pressure application (10 psi for 20 s) of PMA. Open columns, before treatment; dotted columns, after treatment. Values are the mean F SEM of 7–8 neurons from five rats. *P b 0.05, compared to before treatment.
hoc analysis for intergroup comparison. Differences were considered significant at P b 0.05.
3. Results 3.1. Effects of PMA, H-7, and HA1004 on neurons in the AHA of Wistar rats The basal mean arterial pressure was 91 F 2 mm Hg (n = 30) in pentobarbital-anesthetized rats. Individual neurons in the AHA were determined to be angiotensin II-sensitive
Fig. 2. (A–D) Firing responses of angiotensin II-sensitive neurons to pressure application (10 psi for 20 s) of PMA in the AHA of WKY (A and B) and SHR (C and D). (E) Effects of pressure application (10 psi for 20 s) of PMA on the firing rate of angiotensin II-sensitive neurons in the AHA of WKY and SHR. Open columns, before treatment; dotted columns, after treatment. Values are the mean F SEM of 12–14 neurons from eight rats. *P b 0.05, compared to WKY.
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WKY. Application (10 psi for 60 s) of H-7 (10 4 M) onto angiotensin II-sensitive neurons decreased the amplitude of unit firing by 65.8 F 3.5% in all neurons tested in the AHA of SHR (12 neurons from 7 rats), whereas the inhibitor (10 4 M) did not affect it in all neurons tested in the AHA of WKY (10 neurons from 7 rats) (Figs. 3A and B). The decrease in amplitude of firing by H-7 in SHR began around 1 min and lasted for about 3 min after the beginning of H-7 application. The firing rate of angiotensin II-sensitive neurons was not changed by H-7 in both SHR and WKY (Figs. 3C and D). Application (10 psi for 60 s) of HA1004 (10 4 M), a negative control of H-7, onto angiotensin IIsensitive neurons did not affect the firing rate in all neurons tested in the AHA of SHR (10 neurons from 7 rats) (data not shown). Pressure application (10 psi for 5 s) of angiotensin II (10 7 M) onto AHA neurons increased the firing rate in WKY and SHR. In SHR, application of H-7 (10 5 and 10 4 M) onto angiotensin II-sensitive neurons inhibited the increase of firing rate induced by angiotensin II (10 7 M)
in a concentration-dependent manner (Fig. 4A). On the other hand, in WKY, H-7 (10 5 M) did not affect the angiotensin II-induced increase of firing rate, although H-7 (10 4 M) significantly inhibited the increase of firing rate by angiotensin II. Pressure application (10 psi) of vehicle (5–60 s) consistently did not affect the basal amplitude and firing rate (n =21, data not shown), suggesting that the effects found by pressure application of drugs are not due to distortion. Fig. 4B shows the location of recording sites for angiotensin II-sensitive neurons in the AHA of WKY and SHR. Recording sites were located in an area of the AHA. The recording sites for the SHR were similar to those for WKY. 3.3. Effects of 4-aminopyridine on angiotensin II-sensitive neurons in the AHA of WKY and SHR Pressure application (10 psi for 5 s) of 4-aminopyridine (10 4 and 310 4 M), a blocking agent of the transient
Fig. 3. Firing (A and B) and continuous rate-meter recordings (C and D) of angiotensin II-sensitive neurons before, during, and after pressure application (10 psi for 60 s) of H-7 (10 4 M) in the AHA of WKY (A and C) and SHR (B and D). In A and B, applications of H-7 are indicated by underlines. Calibrations in A and B: vertical bars represent 1 mV and horizontal bars represent 1 s.
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angiotensin II-sensitive neurons in the AHA of SHR, whereas this agent did not affect neural activity in the AHA of WKY. In addition, HA1004 did not affect neural activity in the AHA of SHR. These findings are compatible with the idea that intracellular signal transduction mechanisms via PKC are enhanced in angiotensin II-sensitive neurons in the AHA of SHR and that the enhanced intracellular signal transduction mechanisms via PKC are involved in the basal-enhanced neural activity of AHA angiotensin II-sensitive neurons seen in SHR [12]. In a previous study, we demonstrated that tonically released endogenous angiotensins are involved in basal neural activity of angiotensin II-sensitive neurons in the AHA [5]. Thus, it could be expected that exogenously applied angiotensin II-induced activation of AHA neurons is also inhibited by H-7 and that inhibition of neural activation by H-7 is greater in SHR than in WKY. In the present study, indeed, we found that H-7 (10 5 M) inhibited angiotensin II-induced neural activation in SHR but not in WKY. In addition, H-7 (10 4 M) caused a greater inhibition of angiotensin II-induced neural activation in SHR than in WKY, whereas HA1004 (10 4 M) did not affect the angiotensin II-induced neural activation in SHR. In the previous study, both angiotensin II and glutamate caused greater neural activation in the AHA of SHR [12]. Fig. 4. (A) Effects of pressure application (10 psi) of H-7 or HA1004 on the angiotensin II (10 7 M, 10 psi for 5 s)-induced increase of neural firing rate in the AHA of WKY and SHR. Treatment with H-7 or HA1004 was started around 1 min before application of angiotensin II. Open columns, before treatment; dotted columns, after treatment. Values are the mean F SEM of 8–10 neurons from six rats. *P b 0.05, compared to WKY. (B) Diagrams illustrating the location of recording sites for angiotensin II-sensitive neurons in the AHA of WKY and SHR.
potassium current, onto angiotensin II-sensitive neurons increased the firing rate in the AHA of WKY and SHR (Fig. 5). The increases of firing rate by 4-aminopyridine were almost the same in WKY and SHR.
4. Discussion In the present study, the PKC activator PMA caused an increase of neural activity in the AHA of Wistar rats, and the PMA-induced neural activation was inhibited by H-7 but not by HA1004. The inhibitory effect of H-7 on PKC is greater than that of HA 1004, while the inhibitory effects of H-7 on cGMP-dependent protein kinase and cAMP-dependent protein kinase are rather smaller than those of HA 1004 [2,8]. Thus, the results of the present study suggest the involvement of PKC in the PMAinduced neural activation. In the present study, the PMA-induced neural activation of AHA angiotensin II-sensitive neurons was greater in SHR than in WKY. H-7 alone inhibited neural activity of
Fig. 5. (A–D) Firing responses of angiotensin II-sensitive neurons to pressure application (10 psi for 5 s) of 4-aminopyridine (4AP) in the AHA of WKY (A and B) and SHR (C and D). (E) Effects of pressure application (10 psi for 5 s) of 4-aminopyridine (4AP) on the firing rate of angiotensin II-sensitive neurons in the AHA of WKY and SHR. Open columns, WKY; dotted columns, SHR. Values are the mean F SEM of 9–10 neurons from six rats.
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Both agonists increase PKC activity, resulting in an increase of neural activity [20]. Thus, the enhanced intracellular signal transduction mechanisms via PKC in AHA neurons of SHR found in this study could, at least in part, explain the enhanced neural reactivity to both angiotensin II and glutamate. PKC activation causes both the reduction in the delayed rectifier K+ current (I k) and in the transient potassium current (I A), and stimulates calcium current (I Ca), resulting in an increase of neural activity [20,22]. In the present study, blockade of I A by 4-aminopyridine increased the unit firing rate of angiotensin II-sensitive neurons in the AHA of WKY and SHR, and the increase of firing rate by 4-aminopyridine was almost the same in the AHA of both kinds of rats. Thus, we could not find any alteration in intracellular mechanisms via I A in AHA neurons of SHR. In the present study, H-7 inhibited both basal neural activity and angiotensin II-induced neural activation in the AHA of SHR, suggesting that PKC is involved in neural activation caused by both spontaneously released and exogenously applied angiotensins in SHR. In contrast, in the AHA of WKY, H-7 did not affect basal neural activity, whereas it caused a slight but significant inhibition of angiotensin II-induced increase of neural activity. This discrepancy could be explained by assuming that in WKY, PKC is not involved in neural activation caused by a small amount of angiotensins like spontaneously released angiotensins, but this enzyme is involved in neural activation caused by a large amount of angiotensin II like exogenously applied angiotensin II (10 7 M). Clearly, more studies will be needed to clarify why H-7 does not affect basal neural activity in the AHA of WKY. It has been reported that total PKC activity is significantly increased in synaptosomal samples isolated from the forebrain, midbrain, and hind brain of SHR [9]. Tsuda et al. [21] have demonstrated that H-7 reduces the stimulationevoked [3H]-acetylcholine release to a greater extent in superfused slices of striatum from SHR than in those of WKY, suggesting that the PKC system may participate in the central nervous system in hypertension. These findings are compatible with the results of the present study. On the other hand, many papers have demonstrated various alterations other than a defect in PKC in the brain of SHR [19,23]. Thus, it will be next needed to investigate that these alterations found in the brain of SHR are related each other or independent. In summary, this study demonstrates that activation of PKC increases neural activity of angiotensin II-sensitive neurons in the AHA and that this PKC activation-induced increase of neural activity is enhanced in the AHA of SHR. Blockade of PKC activity by H-7 caused a decrease of basal neural activity of angiotensin II-sensitive neurons in the AHA of SHR but not of WKY, suggesting that the enhanced PKC activation effect may be responsible for the enhanced basal neural activity seen in the AHA of SHR.
Acknowledgment This study was supported in part by a Grant-in-Aid for Scientific Research (No. 13672300) from Japan Society for the Promotion of Science.
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