GABAB Receptors Expressed in Human Aortic Endothelial Cells Mediate Intracellular Calcium Concentration Regulation and Endothelial Nitric Oxide Synthase Translocation
GABAB receptors regulate the intracellular Ca2+ concentration ([Ca2+]i) in a number of cells (e.g., retina, airway epithelium and smooth muscle), but whether they are expressed in vascular endothelial cells and similarly regulate the [Ca2+]i is not known. The purpose of this study was to investigate the expression of GABAB receptors, a subclass of receptors to the inhibitory neurotransmitter γ-aminobutyric acid (GABA), in cultured human aortic endothelial cells (HAECs), and to explore if altering receptor activation modified [Ca2+]i and endothelial nitric oxide synthase (eNOS) translocation. Real-time PCR, western blots and immunofluorescence were used to determine the expression of GABAB1 and GABAB2 in cultured HAECs. The effects of GABAB receptors on [Ca2+]i in cultured HAECs were demonstrated using fluo-3. The influence of GABAB receptors on eNOS translocation was assessed by immunocytochemistry. Both GABAB1 and GABAB2 mRNA and protein were expressed in cultured HAECs, and the GABAB1 and GABAB2 proteins were colocated in the cell membrane and cytoplasm. One hundred μM baclofen caused a transient increase of [Ca2+]i and eNOS translocation in cultured HAECs, and the effects were attenuated by pretreatment with the selective GABAB receptor antagonists CGP46381 and CGP55845. GABAB receptors are expressed in HAECs and regulate the [Ca2+]i and eNOS translocation. Cultures of HAECs may be a useful in vitro model for the study of GABAB receptors and vascular biology.
GABAB receptors, a distinct subclass of receptors to the inhibitory neurotransmitter γ-aminobutyric acid (GABA), comprised of two principal heterodimeric subunits GABAB1 and GABAB2 [1–3], are members of the metabotropic receptor family that via Gi/o proteins interact with neuronal inwardly rectifying potassium and voltage-gated calcium channels and when activated mediate slow synaptic inhibition . GABAB receptors are mainly located within the central nervous system and retina [5, 6] and modulate intracellular calcium concentration ([Ca2+]i) in selected neural cells (e.g., chromaffin cells , dopaminergic neurons , and cortical neurons ). GABAB receptors have also been detected in peripheral tissues, including human and guinea pig airway epithelium , human fallopian tube , and human airway smooth muscle , and shown to participate in the [Ca2+]i modulation .
Vascular endothelial cells are principal cells of blood vessels and play crucial roles within the vasculature. Intracellular Ca2+ contributes to vascular endothelium physiology, functions, and disorders such as proliferation , apoptosis , permeability , endothelial nitric oxide synthase (eNOS) activation , injury , and healing . Other neural-transmitter receptor types that are generally thought of as having primarily central neural system locations and functions have been shown to be present within the peripheral vascular endothelium cells and modify the intracellular Ca2+. For example, the muscarinic receptor subtypes 1 and 3 were detected in human vascular endothelial cells . Acetylcholine increases the [Ca2+]i in primary cultured rabbit aortic endothelial cells, and this can be blocked by the selective muscarinic receptor antagonist atropine . β-Adrenoceptors are present in the endothelium of the rabbit coronary artery . Epinephrine induces endothelial Ca2+ influx and thus increases the [Ca2+]i in primary cultured bovine aortic endothelial cells and this can be inhibited by a β2-adrenoceptor antagonist ICI-118551 . 5-Hydroxytryptamine (5-HT)1D, 5-HT2B, and 5-HT4 receptors are expressed in human umbilical vein endothelial cells [24, 25]. 5-HT stimulates Ca2+ uptake and this can be inhibited by 5-HT receptor antagonists .
As the major source of nitric oxide (NO) in vascular endothelial cells , endothelial nitric oxide synthase (eNOS) plays a crucial role within the cardiovascular system. The subcellular location of eNOS contributes to the enzyme functions . In resting endothelial cells, eNOS is mainly located at the cell membrane and cytoplasm, and when stimulated by agonists, it translocates to structures within the cell cytosol close to the nucleus [27, 28]. eNOS translocation can be induced by a variety of agents, some of which stimulate the [Ca2+]i increase in endothelial cells. For example, acetylcholine [21, 29], endothelin-1 [30, 31], platelet-activating factor [29, 32], bradykinin , estrogen , and epicatechin  increase the [Ca2+]i in endothelial cells and induce eNOS translocation. Thus, GABAB receptors regulate the [Ca2+]i in some neural and nonneural cells [7–9, 13]; whether they are expressed in vascular endothelial cells and regulate the [Ca2+]i and eNOS translocation is not clear.
Based on indirect evidence, we hypothesized that GABAB receptors would be expressed in human aortic endothelial cells (HAECs). The purpose of this study was to investigate whether GABAB receptors are expressed in cultured HAECs and regulate the [Ca2+]i and eNOS translocation. If these receptors are present, HAECs could be a useful model for studying a direct role of GABA in vascular regulation.
2.1. Cell Culture
Primary HAECs obtained from the American Type Cell Collection (VA, USA) were cultured in endothelial cell medium (ECM) containing 5% FBS and 1% endothelial cell growth supplement (ScienCell, USA) at 37°C with humidified air and 5% CO2. HAECs of no more than passage 4 were used. The study was carried out in accordance with “The Code of Ethics of the World Medical Association (Declaration of Helsinki)” for experiments involving humans.
2.2. Real-Time PCR
RNA isolation and reverse transcription were performed as previously described . RNA concentration and purity were determined at an optical density ratio of 260 : 280 using a spectrophotometer. Primers for human GABAB1 and GABAB2 were designed to span a region that includes an intron in the genomic sequence for these genes and ordered from Shanghai Biosune Biotechnology Company (Shanghai, China). The primers for GABAB1 were forward 5′-GCCGCTGTGTCCGAATCTGCT-3′ and reverse 5′-CTGCGCGCCGTTCTGAGTGT-3′, and for GABAB2 they were forward 5′-TGGAGGCGTCTGTCCATCCGT-3′ and reverse 5′-GTCTTGCGTCAGCGTGCCCA-3′. SYBR Green real-time PCR and quantitative assays were performed by use of a Real-time PCR Detection System, LightCycler (Roche Applied Science, IN, USA). Denaturation was performed for 10 s at 95°C, annealing for 10 s at 60°C, and extension for 10 s at 72°C. cDNA from the human retinal tissue was used as the positive control and the samples without cDNA were used as the negative control. β-Actin was used as the housekeeping gene. Correct product size (228 bp for GABAB1 and 220 bp for GABAB2) was confirmed by DNA agarose gel, and sequence comparison with target genes was conducted (by Biosune Biotechnology Company, Shanghai). Samples were analyzed in triplicate.
Immunocytochemistry was performed as previously described . Briefly, cells were fixed with 4% paraformaldehyde, blocked with 10% normal donkey serum, and incubated with mouse anti-human GABAB1 antibody (1 : 500; Abcam, USA), goat anti-human GABAB2 antibody (1 : 100; Santa Cruz, CA, USA), or rabbit anti-human eNOS antibody (1 : 100; Sigma, USA) overnight at 4°C. Subsequently, the cells were incubated with donkey anti-mouse secondary antibody (Alexa 568 conjugated; 1 : 1000; Invitrogen, CA, USA), donkey anti-goat secondary antibody (Alexa 488 conjugated; 1 : 1000; Invitrogen, CA, USA), and donkey anti-rabbit secondary antibody (Alexa 488 conjugated; 1 : 1000; Invitrogen, CA, USA) for 1 h at 37°C. A drop of Prolong Gold anti-fade reagent with DAPI (Invitrogen, CA, USA) was added before cell images were acquired by use of a LSM 710 laser confocal microscope (EC Plan-Neofluar 40×/1.30 Oil objective, N.A. 0.55) equipped with ZEN 2009 Light Edition software (Zeiss, Germany).
2.4. Western Blots
Western blots were performed as previously described [35, 36]. HAECs and human retinal tissue (positive control) protein samples were separated by 7.5% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. After 1 h defatted milk block, the membrane was incubated with the anti-GABAB1 antibody (Abcam, 1 : 1000) and anti-GABAB2 antibody (Santa Cruz; 1 : 200) and then with horseradish peroxidase- (HRP-) conjugated secondary antibody (1 : 5000). The bands were visualized by use of Immobilon Western Chemiluminescent HRP Substrate (Millipore, MA, USA).
2.5. Measuring [Ca2+]i
[Ca2+]i was measured as previously described  with minor modification. HAECs were cultured on a glass-covered disc with a concentration of 5 × 105 cells/mL. Two days later, the cells were incubated with 5 μM fluo-3 AM for 20 min in the dark in normal physiological saline solution (N-PSS) that contained (in mM) 140 NaCl, 1 KCl, 1 CaCl2, 1 MgCl2, 10 glucose, and 5 HEPES (pH 7.4) at 37°C. After being rinsed twice with N-PSS, cells were kept in N-PSS for another 10 min, and then the circular discs with HAECs attached were placed on the stage of a confocal microscope. While the images were being acquired, 100 μM agonist baclofen was added to the assay disc at the set time points. To determine the impact of the antagonist, cells were preincubated for 10 min with 1 mM of the GABAB receptor antagonists CGP46381 (Santa Cruz, CA, USA) and CGP55845 (Tocris Bioscience, USA) before 100 μM baclofen (Sigma, MO, USA) was added. Sequences of images were acquired using the laser confocal microscope (LSM 710, Zeiss, Germany) equipped with a 488 nm laser at 5 s intervals. The fluorescence intensity over the HAECs cell body was measured before and after agent application. The fluorescence intensity before addition of agents was considered the baseline fluorescence intensity. The changes of fluorescence intensity after agent application were calculated and analyzed by using ZEN 2009 Light Editin software (Zeiss, Germany). PBS instead of GABAB agents were used as the control.
2.6. eNOS Translocation
eNOS translocation was investigated using immunofluorescence as previously described  with minor modification. To test the effects of the GABAB receptor agonist on eNOS translocation, cells were treated with 100 μM baclofen for 30 min. To determine the impact of the antagonist, cells were preincubated for 10 min with 1 mM of the GABAB receptor antagonists CGP46381 and CGP55845 before 100 μM baclofen was added.
2.7. Statistical Analysis
All data were analyzed using SPSS v16.0 (SPSS Inc., Chicago, IL, USA). Data were acquired from at least 3 independent repeats of the experiments and were expressed as mean ± SD.
Real-time PCR demonstrated the presence of GABAB1, GABAB2, and β-actin mRNA in cultured HAECs and in human retina, but not in the negative control. Results from ethidium bromide-stained agarose gels electrophoresis of the real-time PCR products demonstrated that the specific bands appeared at the position of 228 bp (GABAB1), 220 bp (GABAB2), and 302 bp (β-actin), respectively (Figure 1). Nucleotide sequence analysis confirmed that the sequence of the PCR products is corresponding to the targeted sequence of GABAB1 and GABAB2 mRNA with the primers.
3.1. GABAB1 and GABAB2 Protein Were Detected in Cultured HAECs
Western blots analysis revealed that specific protein bands appeared at approximately 108 kDa (GABAB1), 130 kDa (GABAB2), and 43 kDa (β-actin) (Figure 2(a)). Immunoreactivities to antibodies for GABAB1 and GABAB2 were observed in cultured HAECs. Immunofluorescence was observed in the cell membrane and cytoplasm but not in the nucleus (Figure 2(b)). These data confirmed the expression of GABAB1 and GABAB2 receptor protein in HAECs.
3.2. GABAB Receptors Regulate [Ca2+]i in Cultured HAECs
The GABAB receptor agonist baclofen (100 μM) induced a rapid and transient rise of [Ca2+]i in HAECs. [Ca2+]i reached its peak level in 20–40 s and then gradually declined (Figure 3(a)). The increase of [Ca2+]i induced by 100 μM baclofen was partly (~50%) abolished by preincubation with 1 mM CGP46381 (Figure 3(b)) and was completely inhibited by preincubation with 1 mM CGP55845 (Figure 3(c)). PBS did not cause increase in [Ca2+]i of the cultured HAECs.
3.3. GABAB Receptors Modulate eNOS Translocation in Cultured HAECs
In control HAECs, eNOS immunostaining was predominantly located at the cell membrane and cytoplasm (Figure 4(a)). One hundred μM baclofen incubated HAECs (incubation for 30 min) showed that eNOS immunostaining was changed to intracellular sites close to the nucleus (Figure 4(b)). Preincubation of CGP46381 and CGP55845 for 10 min inhibited 100 μM baclofen induced translocation of eNOS in HAECs (Figures 4(c) and 4(d)).
In the study we found that GABAB1 and GABAB2 mRNA and protein were expressed in cultured HAECs; the two subunits were colocated in the cell membrane and cytoplasm, but neither was located in the nucleus. The GABAB receptor agonist baclofen induced a transient increase of [Ca2+]i and eNOS translocation and the effects were attenuated by the GABAB receptor antagonists CGP46381 and CGP55845. These findings suggest that GABAB receptors are expressed in cultured HAECs and regulate the [Ca2+]i and eNOS translocation.
Muscarinic receptors [20, 21], β-adrenoceptors [22, 23], and 5-HT receptors [24–26] that are primarily located in central neural system are also present within the peripheral vascular endothelial cells and modulate the [Ca2+]i. Here we add GABAB receptors to the list. Whether GABAB receptor functions involving intracellular Ca2+ are similar to those of other neural-transmitters requires further investigation. Possibilities include proliferation , apoptosis , permeability , and eNOS activation .
The [Ca2+]i changes in vascular endothelial cells have been reported to be involved in eNOS activity, which mainly include eNOS translocation and phosphorylation [17, 27]. eNOS translocation from the plasma membrane to subcellular locations contributes to eNOS functions, such as permeability , and thecell membrane-bound and Golgi-bound eNOS are considered to have the ability to release more basal NO than cytosolic eNOS . We found that GABAB receptors modify the eNOS translocation by moving eNOS from the cell membrane and cytoplasm to the cytoplasm closer to the nucleus. It is thus possible that GABAB receptors in HAECs regulate NO production and modify vascular permeability .
GABAB receptors have been reported to be involved in regulating vasculature functions, but the mechanisms are complex. In addition to the central neural system mechanisms [38–40], GABAB receptors directly regulate the vasculature functions via a peripheral mechanism. The GABAB receptor agonist, SKF-97541, induces vasodepression in the feline pulmonary vascular bed and these responses are attenuated after the administration of a GABAB receptor antagonist, saclofen . The GABAB receptor agonist baclofen causes vasodilation in 50% of vessels in the rat retina; the vasodilation can be blocked by the GABAB receptor antagonist 2-hydroxysaclofen . Here we verified that GABAB receptors are expressed and located in cultured HAECs and regulate [Ca2+]i and eNOS translocation. These suggested that vascular endothelial cells would be the potential targets for GABAB receptors directly modulating vascular functions.
In summary, GABAB1 and GABAB2 mRNA and protein were expressed in cultured HAECs and GABAB receptors modified [Ca2+]i and eNOS translocation; this suggests a possible role of GABAB receptors in the mediation of HAECs functions. Further investigation is required regarding which specific HAEC functions (e.g., proliferation, apoptosis, and permeability) GABAB receptors regulate.
|GABAB1:||γ-Aminobutyric acid B receptor subunit 1|
|GABAB2:||γ-Aminobutyric acid B receptor subunit 2|
|HAECs:||Human aortic endothelial cells|
|eNOS:||Endothelial nitric oxide synthase|
|RT-PCR:||Real-time polymerase chain reaction.|
Conflict of Interests
The authors have no conflict of interests to declare.
Xu-Ping Wang and Zhen-Ying Cheng made the same contribution to this paper.
This work was supported by Grant from the National Natural Science Foundation of China (no. 81271038), Grant from the Department of Science and Technology of Shandong Province of China (no. ZR2012HM022) to Zhen-Ying Cheng, Grant from the National Nature Science Foundation of China (no. 80200212), and Grant from the Department of Science and Technology of Shandong Province of China (no. ZR2012HQ029) to Xu-Ping Wang.
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