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. 2023 Mar 6;24(2):e26. doi: 10.4142/jvs.22275

Inhibition of angiotensin converting enzyme increases PKCβI isoform expression via activation of substance P and bradykinin receptors in cultured astrocytes of mice

Jae-Gyun Choi 1,†, Sheu-Ran Choi 2,†, Dong-Wook Kang 1, Hyun Jin Shin 1, Miae Lee 1, Jungmo Hwang 3,✉, Hyun-Woo Kim 1,✉
PMCID: PMC10071283  PMID: 37012034

Abstract

Background

Angiotensin-converting enzyme inhibitor (ACEi) inhibits the catalysis of angiotensin I to angiotensin II and the degradation of substance P (SP) and bradykinin (BK). While the possible relationship between ACEi and SP in nociceptive mice was recently suggested, the effect of ACEi on signal transduction in astrocytes remains unclear.

Objectives

This study examined whether ACE inhibition with captopril or enalapril modulates the levels of SP and BK in primary cultured astrocytes and whether this change modulates PKC isoforms (PKCα, PKCβI, and PKCε) expression in cultured astrocytes.

Methods

Immunocytochemistry and Western blot analysis were performed to examine the changes in the levels of SP and BK and the expression of the PKC isoforms in primary cultured astrocytes, respectively.

Results

The treatment of captopril or enalapril increased the immunoreactivity of SP and BK significantly in glial fibrillary acidic protein-positive cultured astrocytes. These increases were suppressed by a pretreatment with an angiotensin-converting enzyme. In addition, treatment with captopril increased the expression of the PKCβI isoform in cultured astrocytes, while there were no changes in the expression of the PKCα and PKCε isoforms after the captopril treatment. The captopril-induced increased expression of the PKCβI isoform was inhibited by a pretreatment with the neurokinin-1 receptor antagonist, L-733,060, the BK B1 receptor antagonist, R 715, or the BK B2 receptor antagonist, HOE 140.

Conclusions

These results suggest that ACE inhibition with captopril or enalapril increases the levels of SP and BK in cultured astrocytes and that the activation of SP and BK receptors mediates the captopril-induced increase in the expression of the PKCβI isoform.

Keywords: Angiotensins, Neuroglia, Pain, Mice, Protein Kinase C

INTRODUCTION

Angiotensin-converting enzyme (ACE) catalyzes the conversion of angiotensin I to angiotensin II and the degradation of kinins, such as substance P (SP) and bradykinin (BK) [1,2]. The ACE inhibitor (ACEi), captopril, or enalapril is used to treat hypertension and decrease the angiotensin II levels, a potent vasoconstrictor [3]. The ACE inhibitors reduce the risk associated with cardiovascular disease by improving the endothelial function in animal models and humans [4]. While many studies have compared the effects of the sulfhydryl-containing ACEi, captopril, and the non-sulfhydryl-containing ACEi, enalapril in patients with cardiovascular disease, captopril and enalapril generally had equivalent effects on the quality of life in patients with hypertension [3]. The clinical use of the ACEi is closely involved in an antihypertensive effect by blocking renin-angiotensin system, whereas it is unclear if ACE inhibition with captopril or enalapril modulates the expression of SP and BK in astrocytes directly by inhibiting their degradation.

The inhibition of ACE with captopril or enalapril induces mechanical allodynia, which is mediated by the SP-induced activation of the neurokinin-1 receptor (NK-1 receptor) in the spinal cord of mice [5]. The effects of ACEi might be closely related to SP/NK-1 receptor-mediated pathways on nociceptive signaling in the central nervous system. Neurons and astrocytes carry receptors for SP on their surfaces, and the activation of these receptors results in neuroinflammation, which is the most common factor causing neurological disorders, such as traumatic brain injuries, stroke, and Alzheimer’s disease [6,7,8]. Furthermore, BK induces a transient increase in the intracellular Ca2+ concentration in cultured astrocytes via BK B1 and B2 receptors [9] and stimulates astrocytes to release cytokines and glutamate [10,11]. Although SP and BK can affect the function of astrocytes by binding to their receptors, it is unclear if SP or BK receptor-mediated signaling mediates the ACE inhibition-induced changes in astrocytes.

Protein kinase C (PKC) mediates diverse cellular signaling pathways contributing to the functional changes in astrocytes [12]. Although it has been suggested that SP- and BK-mediated receptor signaling is involved in the actions of various PKC isoforms [13,14,15], the effects of SP and BK on protein expression of PKC isoforms in astrocytes remain unclear. Hence, experiments were conducted to determine if the 1) inhibition of ACE with the ACE inhibitor, captopril or enalapril increases the levels of SP and BK in cultured astrocytes, 2) these captopril- or enalapril-induced changes are reversed by the pretreatment with ACE, 3) inhibition of ACE with captopril modulates the expression of PKC isoforms (PKCα, PKCβI, and PKCε) in cultured astrocytes, and, 4) pretreatment with the NK-1 receptor antagonist, L-733,060, the BK B1 receptor antagonist, R 715, or the BK B2 receptor antagonist, HOE 140 inhibits the captopril-induced changes in the expression of the PKC isoforms in cultured astrocytes.

MATERIALS AND METHODS

Preparation of primary astrocyte cultures

As described previously [16,17,18], astrocyte cultures were produced from the cerebral cortices of postnatal two-day-old ICR (Institute of Cancer Research) mice (Samtaco Bio Inc., Korea). The brains of mice pups were removed, and the cerebral cortices were collected. The astrocytes were recovered by repeatedly removing isolated cells from brain tissues after removing the meninges. The T75 culture flask was kept at 37°C in a 95% air/5% CO2 incubator for two–three weeks in minimal essential medium (MEM, CAS No. C8856-5G, Gibco by life technologies, USA). The media were changed twice a week. For 24 h, the T75 flasks were vigorously shaken to eliminate oligodendrocytes and microglia.

Astrocyte cells were split into 12 well plates (1 × 105 cells/well both with and without cover glass) and cultured for three–four days before use. The Animal Care and Use Committee at the Chungnam National University (approval No. 202209A-CNU-156) approved this experiment.

Drugs

Captopril (CAS No. C8856-5G, an angiotensin-converting enzyme inhibitor, 1 or 10 mg/mL, incubated for 24 h), Enalapril maleate (CAS No. PHR1289-1G, an angiotensin-converting enzyme inhibitor, 1 or 10 mg/mL, incubated for 24 h), and Angiotensin-converting enzyme from rabbit lung (CAS No. A6778, 35 μg/mL, treated 1 h before ACEi administration) were purchased from Sigma–Aldrich (USA). L-733,060 hydrochloride (CAS No. 148687-76-7, a potent neurokinin-1 receptor antagonist, 3 μg/mL, treated one hour before ACEi administration), R 715 (CAS No. 185052-09-9, a potent and selective BK B1 receptor antagonist, 3 μg/mL, treated 1 h before ACEi administration), and HOE 140 (CAS No. 130308-48-4, a potent and selective BK B2 receptor antagonist, 3 μg/mL, treated 1 h before ACEi administration) were purchased from Tocris Cookson Ltd. (UK). All drug doses were based on preliminary data and did not cause abnormal changes in cultured cells. All drugs were dissolved in physiological saline.

Western blot analysis

Western blot analysis was used to examine the changes in the protein expression of PKC isoforms in primary cultured astrocytes, as described previously [19,20]. Cultured astrocytes were transfected with the ACE inhibitor, captopril (1 mg/mL, n = 5) for 24 h. In other experiments, astrocytes were pretreated with L-733,060 (3 μg/mL, n = 5), R 715 (3 μg/mL, n = 5), or HOE 140 (3 μg/mL, n = 5) for 1 h before captopril administration and transfected with captopril (1 mg/mL) for 24 h. The astrocytes were collected and lysed with lysis buffer (1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.05 M Tris-HCl pH 8) containing protease and phosphatase inhibitor for 30 min on ice, and then collected for bicinchoninic acid protein assay kit (Thermo Fisher Scientific, USA) protein concentration assurance. On a 12% SDS-PAGE gel, equal protein samples (25 mg) were loaded and transferred to a nitrocellulose membrane. The membrane was washed in Tris-buffered saline (10 mM Tris, 150 mM NaCl) containing 0.1% Tween 20 (TBST) and blocked with TBST containing 5% bovine serum albumin Fraction V (Roche, Switzerland). The primary antibodies against the rabbit anti- PKCα antibody (CAS No. sc-208, 1:1,000, Santa Cruz Biotechnology, USA), rabbit anti- PKCβI antibody (CAS No. sc-209, 1:1,000, Santa Cruz Biotechnology), rabbit anti- PKCε antibody (CAS No. sc-214, 1:1000, Santa Cruz Biotechnology), and rabbit anti-GAPDH antibody (CAS No. sc-25778, 1:2000, Santa Cruz Biotechnology) were incubated overnight with the membranes at 4°C. Subsequently, the samples were exposed to secondary anti-rabbit horseradish peroxidase-conjugated antibodies. The chemiluminescent signals were obtained using the Super Signal West Pico or Femto Substrate (Pierce Biotechnology, USA), and the scanned images were analyzed using Image J (Version 1.52a). Using LabWorks software (UVP, USA), the band intensity of the targeted bands was recorded in an Epichem 3 Darkroom and normalized against the contrasting GAPDH loading control bands. The experiment was repeated three times independently.

Immunocytochemistry and image analysis

Immunocytochemistry was performed to look at the changes within the levels of SP and BK in cultured astrocytes, as described elsewhere [18]. Primary astrocytes grown on cover glass were fixed in 100% methanol at −20°C for 10 min. The cultured cells were incubated with a blocking solution and 0.1% Tween-20 in phosphate-buffered saline (PBS) for 1 hour following three PBS washes. The cells were incubated with primary antibodies overnight at 4°C after three additional PBS washes. The primary antibodies against mouse anti-glial fibrillary acidic protein (GFAP) antibody (CAS No. MAB360, a marker of astrocyte, 1:1,000, USA), rabbit anti-GFAP antibody (CAS No. Z0334, a marker of astrocyte, 1:1000, Dako Products, USA), mouse anti-SP antibody (CAS No. ab14184, 1:500, Abcam, Korea), and rabbit anti-BK antibody (CAS No. PA5-79571, 1:500, Thermo Fisher Scientific, USA). After three further washes to remove the remaining primary antibody, the cultures were incubated for 2 h at room temperature with Cy3-conjugated anti-mouse antibody (CAS No. 715-165-150, 1:200, Jackson ImmunoResearch LABORATORIES, INC., USA) and FITC-conjugated anti-rabbit antibody (CAS No. 711-095-152, 1:200, Jackson ImmunoResearch LABORATORIES, INC.) before being mounted serially (vectashield, Vecta) onto silane-coated slides. The experiment was repeated three times independently.

The dimensional structure and morphology of the cell were analyzed using Image J. The level of colocalization of SP or BK with GFAP was determined by counting the number of SP or BK-immunoreactive cells and expressing them as the percentage of SP or BK-immunoreactive GFAP-positive cells relative to the total number of GFAP-positive cells visualized in cultured astrocytes.

Statistical analysis

The data were presented as the mean ± SEM. GraphPad Prism Software (Version 6.01, USA) was used for the statistical analysis. One-way ANOVA was used to analyze the differences between the experimental groups, and then Bonferroni’s multiple comparisons test was performed for post hoc analysis. p-values < 0.05 were considered significant.

RESULTS

Captopril or enalapril treatment increases the SP immunoreactivity in primary cultured astrocytes

Immunocytochemistry was performed using a specific antibody against SP or GFAP at 24 h after captopril or enalapril treatment to determine if ACE inhibition with captopril or enalapril increases the levels of SP in cultured astrocytes. Immunocytochemistry revealed that SP immunoreactivity was increased after captopril (Fig. 1A; 1 or 10 mg/mL, n = 7) or enalapril (Fig. 1B; 1 or 10 mg/mL, n = 7) treatment in GFAP-positive cultured astrocytes when compared with the vehicle-treated group (Fig. 1C; p < 0.001 vs. vehicle control group). The effect of ACE treatment on the captopril- and enalapril-increased levels of SP in cultured astrocytes was examined to determine if the increased SP production is caused by the captopril- or enalapril-induced ACE inhibition. Pretreatment with ACE (35 µg/mL, n = 7) reduced the SP immunoreactivity that was increased by the captopril or enalapril treatment in GFAP-positive astrocytes (Fig. 1C; p < 0.001 vs. captopril- or enalapril-treated group).

Fig. 1. Inhibition of ACE increases the levels of substance P in cultured astrocytes. (A and B) Representative images show the changes in SP (red) and GFAP (green) immunoreactivity after treatment with captopril (A) or enalapril (B) and its attenuation by ACE in primary cultured astrocytes. (C) The graph shows that SP immunoreactivity in GFAP-positive astrocytes is increased by a treatment with captopril (1 or 10 mg/mL) or enalapril (1 or 10 mg/mL), which was inhibited by the pretreatment with ACE (35 µg/mL).

Fig. 1

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SP, substance P; GFAP, glial fibrillary acidic protein; ACE, angiotensin-converting enzyme.

***p < 0.001 vs. vehicle control group, ###p < 0.001 vs. captopril- or enalapril-treated group.

BK immunoreactivity is elevated in primary cultured astrocytes following the captopril or enalapril treatment

The effects of ACE inhibition with captopril or enalapril on the levels of BK in cultured astrocytes were examined using immunocytochemistry. BK immunoreactivity was increased after captopril (Fig. 2A; 1 or 10 mg/mL, n = 7) or enalapril (Fig. 2B; 1 or 10 mg/mL, n = 7) treatment in GFAP-positive cultured astrocytes compared to the vehicle-treated group (Fig. 2C; p < 0.001 vs. vehicle control group). The effect of ACE treatment on the captopril- and enalapril-induced increase in the levels of BK in cultured astrocytes was examined to determine if the increased BK production is caused by the captopril- or enalapril-induced ACE inhibition. Pretreatment with ACE (35 µg/mL, n = 7) reduced the BK immunoreactivity increased by captopril or enalapril treatment in GFAP-positive astrocytes (Fig. 2C; p < 0.001 vs. captopril- or enalapril-treated group).

Fig. 2. Inhibition of ACE increases the levels of bradykinin in cultured astrocytes. (A and B) Representative images show the changes in bradykinin (green) and GFAP (red) immunoreactivity after treatment with captopril (A) or enalapril (B) and its attenuation by ACE in primary cultured astrocytes. (C) The graph shows that bradykinin immunoreactivity in GFAP-positive astrocytes is increased by treatment with captopril (1 or 10 mg/mL) or enalapril (1 or 10 mg/mL), and inhibited by a pretreatment with ACE (35 µg/mL).

Fig. 2

Open in a new tab

BK, bradykinin; GFAP, glial fibrillary acidic protein; ACE, angiotensin-converting enzyme.

***p < 0.001 vs. vehicle control group, ###p < 0.001 vs. captopril- or enalapril-treated group.

Treatment with captopril increases the expression of the PKCβI isoform in primary cultured astrocytes via the activation of NK-1, BK B1, and BK B2 receptors

The effect of captopril (1 mg/mL, n = 5) on the protein expression of PKC isoforms (PKCα, PKCβI, and PKCε) in primary cultured astrocytes was next examined because PKC is a vital serine- and threonine-specific protein kinase to activate diverse cellular signaling pathways. Fig. 3A presents representative immunoblots of PKC isoforms, and GAPDH obtained from Western blot assay. Treatment with captopril did not affect the expression of the PKCα isoform compared to the vehicle-treated group (Fig. 3B). In contrast, the expression of the PKCβI isoform was statistically increased by the treatment with captopril (Fig. 3C; p < 0.001 vs. vehicle control group). The vehicle control and captopril-treated groups showed similar expression of the PKCε isoform (Fig. 3D).

Fig. 3. Effect of captopril on the expression of PKC isoforms in primary cultured astrocytes. (A) Representative immunoblots of the expression of PKCα, PKCβI, and PKCε isoforms are shown. (B) Captopril treatment (1 mg/mL) did not affect the expression of PKCα isoform in primary cultured astrocytes. (C) The expression of PKCβI isoform was increased by a treatment with captopril, and the pretreatment suppressed this increase with L-733,060 (3 µg/mL), R 715 (3 µg/mL), or HOE 140 (3 µg/mL). (D) Captopril treatment did not affect the expression of PKCε isoform in primary cultured astrocytes.

Fig. 3

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***p < 0.001 vs. vehicle control group, #p < 0.1, ###p < 0.001 vs. captopril-treated group.

The effects of the NK-1 or BK receptor antagonist on the captopril-induced changes in PKC isoforms were examined to verify whether the changes induced by captopril treatment resulted from the direct activation of SP or BK receptor. NK-1 receptor antagonism with L-733,060 (3 µg/mL, n = 5) significantly suppressed the captopril-induced increased expression of PKCβI isoform and the basal expression of PKCα isoform when compared to the captopril-treated group (Fig. 3B and C; p < 0.1, p < 0.001 vs. captopril-treated group). Pretreatment with the BK B1 receptor antagonist, R 715 (3 µg/mL, n = 5), or the BK B2 receptor antagonist, HOE 140 (3 µg/mL, n = 5) inhibited the captopril-induced increased expression of PKCβI isoform (Fig. 3C; p < 0.001 vs. captopril-treated group).

DISCUSSION

Emerging evidence suggests that ACE is expressed in the nervous system and acts as a local putative receptor for angiotensin [21]. ACE is expressed in the lumbar dorsal root ganglion and the superficial dorsal horn region of the lumbar spinal cord in mice [5]. The present study showed that captopril and enalapril increased the levels of SP and BK significantly in cultured astrocytes, which were reversed by pretreatment with exogenous ACE. These results showed that direct inhibition of ACE in cultured astrocytes with captopril and enalapril prevents the degradation of SP and BK. The levels of SP and BK may have been restored by the treatment with exogenous ACE in cultured astrocytes because exogenous ACE can catalyze the degradation of SP and BK. Interestingly, there was no significant difference in the levels of SP and BK between captopril- and enalapril-treated groups. These results suggest that captopril and enalapril have similar inhibitory effects on the ACE-mediated degradation of SP and BK in primary cultured astrocytes from mouse brains, even though these two drugs are included in different drug classes.

SP and BK are mediators of the inflammatory response [22]. Hence, abnormal increases in the levels of SP and BK could bring undesirable effects on the nervous system. Moreover, SP stimulates the formation of arachidonate-derived proinflammatory and immunoregulatory compounds, prostaglandin E and thromboxane B2, and the production of inflammatory cytokines, interleukin-6 and interleukin-8, in human astrocytoma cell line [7,23]. In the central nervous system, inflammatory cytokines released from SP-stimulated astrocytes are closely related to the pathophysiology of inflammatory neurodegenerative disorders [6,23]. SP-immunoreactive astrocytes are found in multiple sclerosis plaque, suggesting a relationship between the SP and pathological changes in astrocytes under the disease state [24]. In addition, SP potentiates glutamate-induced ATP release from spinal cord astrocytes [14], and BK induces glutamate release from astrocytes, which mediates astrocyte-to-neuron signaling in the brain during inflammation [11]. These results indicate that the captopril- and enalapril-induced increased SP and BK in astrocytes may play a role in astrocyte-neuron communication under the pathophysiological conditions of the nervous system.

In the present study, the expression of the PKCβI isoform was increased significantly by a captopril treatment, which was reduced partially by blocking the BK B2 receptor and suppressed by blocking the BK B1 or NK-1 receptor. When SP binds to the NK-1R in astrocytes, this leads to G-protein activation of phospholipase C, which increases inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) levels in the cells [23]. Miyano et al. suggested that stimulation of the NK-1 receptors in spinal astrocytes of rats induced the release of Ca2+ from IP3-sensitive intracellular Ca2+ stores and extracellular Ca2+ influx through canonical transient receptor potential 3 channel [25]. BK receptors, B1 and B2 receptors, are also G-protein-coupled receptors and increase intracellular Ca2+ levels in sensory neurons and glioma cells [26,27,28]. Thus, activation of the NK-1 and BK B1 receptors is actively involved in modulating PKCβI expression through G-protein- or Ca2+-mediated signaling in astrocytes. Furthermore, antagonism of the NK-1 receptor significantly reduced the expression of PKCβI and the PKCα and PKCε isoforms compared to those of control groups, suggesting that NK-1 receptor stimulation is essential for the maintenance of the basal expression of PKCβI, PKCα, and PKCε isoforms.

In contrast to the PKCβI isoform, neither PKCα nor PKCε isoform was increased in the primary cultured astrocytes by ACE inhibition with captopril. The hormonal and physiological changes in the environmental conditions modulate PKC isoform gene expression in the heart [29]. Iron is an essential element required for cell proliferation and is involved in regulating PKCβ but not PKCα gene expression in various cultured cell lines [30]. Although the mechanisms responsible for modulating the expression of the PKC isoforms in astrocytes are unknown, the present results suggest that the role of PKCβI isoform may be specific in the functions of astrocytes.

PKC isoforms have been divided into three groups: classical (α, βI, βII, and γ), novel (δ, ε, η, and ), and atypical (ξ and ι/λ) [31,32,33]. PKCβ is a classical isoform activated by Ca2+ and DAG. In the present study, the captopril treatment increased the expression of the PKCβI isoform in cultured astrocytes, while it is unclear if the activity of the PKCβI isoform was also increased by the treatment with captopril. The PKCβI isoform might mediate the diverse actions of astrocytes through the phosphorylation of serine and threonine residues because the activation of SP and BK receptors may provide Ca2+ and DAG that are needed to activate classical PKC isoforms. PKCβ is important for B cell activation and is linked functionally to receptor-mediated survival signaling for NF-κB activation [32]. The activation of PKCβ, particularly the PKCβI isoform, is involved in an increased mRNA expression of transforming growth factor-β in glomeruli of diabetic rats [34]. Hama et al. [35] suggested that local contact with astrocytes promotes excitatory synaptogenesis in neurons with PKC signaling. In addition, the activation of PKCβI is involved in the morphological changes in astrocytes [36]. Although astrocytes play a key role in the nervous system activation under the pathophysiological states, further studies will be needed to investigate the role of the PKCβI isoform that may be increased by the activation of SP and BK receptors in astrocytes.

In conclusion, the present study showed that the captopril- and enalapril-induced inhibition of ACE increases the levels of SP and BK in cultured astrocytes. In addition, treatment with captopril increases the expression of the PKCβI isoform via the activation of NK-1, BK B1, or BK B2 receptors in cultured astrocytes. These findings also indicate that the angiotensin-converting enzyme expressed in astrocytes plays a vital role in controlling the PKCβI-mediated signaling pathways by catalyzing the degradation of both SP and BK. This study suggests the possibility that the blockade of SP and BK receptors may alleviate the effect of ACE inhibition on astrocytes located in the central nervous system.

Footnotes

Funding: This research was supported by Chungnam National University, Chungnam National University Hospital Research Fund (2021) and the National Research Foundation of Korea grant funded by the Korea government (NRF-2021R1F1A1062509).

Conflict of Interest: The authors declare no conflicts of interest.

Author Contributions:
  • Conceptualization: Choi JG, Choi SR, Hwang J, Kim HW.
  • Data curation: Choi JG, Choi SR.
  • Formal analysis: Choi JG, Choi SR.
  • Funding acquisition: Hwang J, Kim HW.
  • Investigation: Choi JG, Kang DW, Shin HJ, Lee M.
  • Methodology: Choi JG, Kang DW, Shin HJ, Lee M.
  • Project administration: Hwang J, Kim HW.
  • Resources: Hwang J, Kim HW.
  • Supervision: Hwang J, Kim HW.
  • Validation: Kang DW, Shin HJ, Lee M.
  • Visualization: Choi JG, Choi SR, Hwang J, Kim HW.
  • Writing - original draft: Choi JG, Choi SR.
  • Writing - review & editing: Hwang J, Kim HW.

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