Activation of Inward Rectifier K+ Channel 2.1 by PDGF-BB in Rat Vascular Smooth Muscle Cells through Protein Kinase A

Platelet-derived growth factor-BB (PDGF-BB) can induce the proliferation, migration, and phenotypic modulation of vascular smooth muscle cells (VSMCs). We used patch clamp methods to study the effects of PDGF-BB on inward rectifier K+ channel 2.1 (Kir2.1) channels in rat thoracic aorta VSMCs (RASMCs). PDGF-BB (25 ng/mL) increased Kir2.x currents (−11.81 ± 2.47 pA/pF, P < 0.05 vs. CON, n = 10). Ba2+(50 μM) decreased Kir2.x currents (−2.13 ± 0.23 pA/pF, P < 0.05 vs. CON, n = 10), which were promoted by PDGF-BB (−6.98 ± 1.03 pA/pF). PDGF-BB specifically activates Kir2.1 but not Kir2.2 and Kir2.3 channels in HEK-293 cells. The PDGF-BB-induced stimulation of Kir2.1 currents was blocked by the PDGF-BB receptor β (PDGF-BBRβ) inhibitor AG1295 and was not affected by the PDGF-BBRα inhibitor AG1296. The PDGF-BB-induced stimulation of Kir2.1 currents was blocked by the protein kinase A inhibitor Rp-8-CPT-cAMPs; however, the antagonist of protein kinase B (GSK690693) had marginal effects on current activity. The PDGF-BB-induced stimulation of Kir2.1 currents was enhanced by forskolin, an adenylyl cyclase (AC) activator, and was blocked by the AC inhibitor SQ22536. We conclude that PDGF-BB increases Kir2.1 currents via PDGF-BBRβ through activation of cAMP-PKA signaling in RASMCs.


Introduction
Platelet-derived growth factor-BB (PDGF-BB) is considered the major stimulant for vascular smooth muscle cell (VSMC) transition from a contractile state (also termed differentiated) to a synthetic state (also termed dedifferentiated) [1][2][3].VSMC phenotype switching plays a critical role in the pathophysiology of arterial remodeling in many vascular diseases including hypertension, atherosclerosis, and restenosis after angioplasty [4]. However, the molecular mechanisms underlying PDGF-BB-induced VSMC phenotype switching are not entirely clear.
Inward rectifier K + channel 2.1 (Kir2.1), encoded by the KCNJ2 gene, is a member of the classic inwardly rectifying potassium channel family (Kir2.x). The channels of this family are constitutively active and exhibit strong inward rectification [10]. Kir2.1 plays biophysical roles in coronary, cerebral, and basilar arterial VSMCs with adenosine increasing Kir2.1 currents via the A3 receptor through activation of PKA in rabbit coronary arterial VSMCs [11][12][13]. In a previous study, we demonstrated that knockdown of Kir2.1 gene expression inhibits PDGF-BB-induced proliferation, migration, the rat VSMC phenotype, and postballoon injury intimal hyperplasia [14]. However, the detailed molecular mechanisms, particularly the electrophysiological regulation, have not been fully explored.
To address these questions, we studied the regulatory mechanisms of Kir2.1 by PDGF-BB in rat thoracic aorta VSMCs (RASMC) using the whole-cell patch clamp technique and Western blot analysis.

Animals and Ethical
Considerations. Male Sprague-Dawley rats (150-180 g) were obtained from the Southeast University Animal Center. The animals were housed in a vivarium under controlled photocycle (12 h light/12 h dark) and temperature (22-25°C) conditions with free access to food and water. All procedures in the present study were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Care of Experimental Animals Committee of Southeast University (approval ID: SYXK-2015.4171).

Cell Preparation.
The male Sprague-Dawley rats (150-180 g) were anesthetized with 10% chloral hydrate (3000 mg/kg, intraperitoneally) and exhibited no signs of peritonitis, pain, or discomfort following administration of 10% chloral hydrate. The Sprague-Dawley rats were anesthetized after 2-3 minutes; rats were euthanized with 6-9% isoflurane. If the Sprague-Dawley rats' hearts have not been beating, non-spontaneous breathing lasts 2-3 minutes, and there is no blinking reflex, the rats are considered dead. Rats were euthanized with an overdose of isoflurane, and RASMCs were isolated from the intimal-medial layers of the thoracic aorta as described [15]. Primary cells were cultured in Dulbecco's modified Eagle medium (DMEM)/F12 supplemented with 20% fetal bovine serum (FBS; Gibco BRL, Gaithersburg, MD) and 100 μg/mL streptomycin-penicillin in an incubator at 37°C with 5% CO 2 . After passaging, RASMCs were cultured in DMEM/F12 with 10% FBS. Cells in the second to third passage were used for all experiments to prevent cell dedifferentiation.

Drugs.
All pharmacological compounds were prepared as aqueous or dimethyl sulfoxide stock solutions of >1,000 times the concentration used during the experiment. Recombinant mouse PDGF-BB was purchased from BioLegend (San Diego, CA, USA). AG1296, AG1295, GSK690693, and forskolin were purchased from Sigma (St. Louis, MO, USA). Rp-8-CPT-cAMPs and SQ22536 were purchased from BIOLOG Life Science Institute (La Jolla, CA, USA).

Cell
Transfection of Kir2.x. Transfection was performed as previously described [5].
2.8. Statistics. All experiments were repeated at least three times. The results are presented as the mean ± SEM. Statistical analyses were performed using one-way ANOVAs. LSD test was used for comparison between ≤3 sets of data as a post hoc test. Bonferroni test was used for comparison between >3 sets of data. The differences between two groups were considered statistically significant at P < 0:05. SPSS 19.0 statistical software (SPSS Inc., Chicago IL, USA) was used for data analyses.
To gain insight into the mechanism of PDGF-BB action on the Kir2.x channel, we tested the effects of PDGF-BB on the properties of a single Kir2.x channel expressed in RASMCs.
A single-channel current was recorded with a pipette voltage of 70 mV using the excised inside-out configuration and then was identified by its unitary conductance (13-

Discussion
Our observations revealed that PDGF-BB activated the Kir2.1 channel, the PDGF-BB-induced activation of the Kir2.1 channel was mediated by activation of the PDGFβ receptor, and the response occurred via the activation of AC and PKA. Regulation of the proliferation, migration, and phenotypic modulation in VSMCs by PDGF-BB has been studied extensively [19]. Ion channel status is associated with several occlusive vascular diseases involving VSMC phenotypic modulation. For example, the switch toward intermediateconductance Ca 2+ -activated K + channel (BK Ca ) expression may promote excessive neointimal VSMC proliferation, and dysfunction of K + channels is linked to pulmonary arterial  BioMed Research International remodeling [20,21]. Reduction in the L-type Ca 2+ channel α1 subunit (Cav1.2) has been observed in rat aortic VSMCs during dedifferentiation and after balloon injury [22,23]. Decreased K + channel activity causes depolarization of the E m and subsequently elevates free Ca 2+ concentration in the cytoplasm via opening of Cav channels, which is required for VSMC proliferation and remodeling [21]. The phenotype-dependent plasticity of Kir channels may have relevance to vascular remodeling [24]. In VSMCs, only Kir2.1 has been identified [13]. Blood vessels in Kir2.2 knockout mice dilate normally in response to high K + stimulation but not in vessels from Kir2.1 knockout mice [25]. Therefore, Kir2.1 seems to be a main subunit in the formation of classic Kir currents in these cells. Recently, we reported that PDGF-BB promotes expression of Kir2.1 channel protein in RASMCs. Electrophysiological studies demonstrated that the functional upregulation of BK Ca is required for PDGF-BB-induced coronary SMC phenotypic modulation and migration [26]. In this study, we found that PDGF-BB increases Kir2.1 channel currents in RASMCs (Figure 2(d)). The single-channel results also revealed that PDGF-BB enhanced the NPo of the Kir2.1 channel (Figure 1(d)).
To date, four distinct types of K + channels have been identified in VSMCs: voltage-gated K + (Kv) channels, ATPsensitive potassium (K ATP ) channels, BK Ca channels, and Kir channels [14]. Steady-state modulation of Kv channels in rat arterial SMC by cAMP-dependent PKA and Kv7.5 channel activity can influence RASMCs via cAMP/PKA activation [27,28]. Allicin activated K ATP channels in rat mesenteric arteries through PKA, and isoflurane activates PKA in rat VSMCs, which in turn activates K ATP channels [29,30]. Baicalin promoted relaxation of mesenteric by activation of BK Ca through stimulation of the cAMP/PKA pathway [31]. Adenosine increased Kir currents via G protein-coupled receptor A3 through the activation of PKA in rabbit coronary arterial SMC [11]. PKA/cAMP may enhance VSMC phenotype switching. This research indicated that PDGF-BB increased Kir2.1 currents via PDGF-BBRβ through the activation of PKA in RASMCs. However, Akt had no effects on Kir2.1 currents of PDGF-induced RASMCs. The single-channel research showed that AC increased NPo of Kir2.1 channel currents ( Figure 5(d)).
PDGF-BB works by activating the PDGF-BBRβ which activates AC, increasing cAMP and activating PKA. If this is the case, then applying PDGF-BB to inside-out patches should have no effect, yet Figure 1(e) suggests an immediate effect. The underlying mechanisms of the phenomenon maybe that PDGF-BB works by activating the PGDF-BBβ receptor which activates AC, increasing cAMP and activating PKA, and activates the Kir2.1 channel in the other mechanism immediately.
The abnormal proliferation, migration, and phenotypic modulation of VSMCs are critical processes in atherosclerosis and restenosis [32,33]. PDGF-BB can initiate a multitude of biological effects through the activation of intracellular signal transduction pathways that contribute to VSMC proliferation, migration, and phenotypic modulation [34]. Therefore, the inhibition of PDGF-induced VSMC proliferation, migration, and phenotypic modulation may represent an important point of therapeutic intervention in atherosclerosis and restenosis.

Conclusion
In conclusion, our study demonstrated that the activation of Kir2.1 channels by PDGF-BB results from the activation of the cAMP-PKA pathway via the PDGF-BBβ receptor in RASMCs. Therefore, Kir2.1 may be a potential candidate for preventing or treating vascular diseases relevant to VSMC proliferation, migration, and phenotypic modulation.

Data Availability
The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.