Receptor subtype involved in α₁-adrenergic receptor-mediated Ca²⁺ signaling in cardiomyocytes¹

Introduction

The α₁-adrenergic receptors (α₁-AR) play a key role in the modulation of sympathetic nervous system activity as well as a site of action for therapeutic agents, such as antihypertensive drugs. Three subtypes of the α₁-AR, α_1A-AR, α_1B-AR, and α_1D-AR have been cloned and each has specific tissue distribution and pharmacological properties^[1,2]. All subtypes of the α₁-AR are Gq protein-coupled receptors, which upon activation, catalyze the cleavage of poly-phosphoinositide into dual signaling molecules, inositol 1,4,5-trisphosphate (IP₃), and diacylglycerol via the activation of phospholipase C. IP₃ leads to the opening of IP₃ receptor channels at the endoplasmic/sarcoplasmic reticulum, and subsequently the release of intracellular Ca²⁺, while the activation of protein kinase C is the downstream signaling pathway for diacylglycerol^[3,4]. Through these signal transduction pathways, the intracellular responses upon α₁-AR stimulation are induced.

Accumulating studies have indicated that the α₁-AR system appears to play a role in cardiac growth, cardiac contrac-tion, and cardiac automaticity in physiological condition^[4–6], as well as in cardiac pathological processes, such as arrhythmo-genesis or cardiac adaptation to various situations^[5,7,8]. Although the exact underlying mechanisms have not been conclusively determined, the increase in intracellular Ca²⁺ signaling, a common event seen in α₁-AR stimulation, is considered to be a primary signaling pathway initiating acute as well as chronic cardiac function modulations by the α₁-AR^[2,7–11]. For instance, the α₁-AR-mediated mobilization of Ca²⁺ from the sarcoplasmic reticulum contributes significantly to excitation–contraction coupling in atrial myocytes, and causes arrhythmogenic intracellular Ca²⁺ oscillations in the ischemic heart^[7,9,10]. Additionally, α₁-AR-mediated Ca²⁺ signaling is essential for the activation of calmodulin-dependent protein kinase II and nuclear factor of activated T cells, both of which signal a hypertrophic program of cardiac gene expression^[8,12,13].

All 3 subtypes of α₁-AR have been detected at the levels of messenger RNA as well as protein in the heart^[14,15]. How-ever, the subtype of the receptor in the mediation of cardiac function is not clear. Many studies have suggested that the α_1A- AR and α_1B-AR appear to play major roles in the heart^[3,6,15,16]. More recently however, the α_1A-AR has been demonstrated to sufficiently induce cardiac arrhythmias and hypertrophy, while the α_1B-AR seems less important^[7,17,18]. Furthermore, the avtivation of α_1B-AR even inhibits α_1A-AR mediated cardiac remodeling^[19], but plays a crucial role in the generation of dilated cardiomyopathy^[16]. As an increase in intracellular Ca²⁺ is the primary signaling transduction pathway for α₁-AR-mediated cardiac function^[2,7–11], and the subtype involved is unclear, in this study we intended to identify the subtype of the α₁-AR involved in mediating intracellular Ca²⁺ signaling by using neonatal rat ventricular myocytes (NRVM), which express all 3 α₁-AR subtypes^[14,15] and respond to α₁-AR stimulation markedly in the profiles of intracellular Ca²⁺ signaling and hypertrophic growth^[8,20,21].

Material and methods

Isolation and culture of cardiomyocytes NRVM were isolated from 1–2-d-old Sprague-Dawley rats by enzymatic digestion with 0.1% trypsin and 0.03% collegenase, as previously described^[20]. Then the myocytes were plated onto laminin-coated, 35 mm dishes at a density of 0.5×10³–0.8×10³ cells/mm² and cultured for 42 h in Dulbecco's modifi-ed Eagle’s medium (DMEM) and Medium 199 (4:1) containing 10% fetal bovine serum, 4 mmol/L L-glutamine, 100 units/mL penicillin/streptomycin, and 0.1 mmol/L 5-bromo-2-deoxyuridine to inhibit fibroblast proliferation. Before use, the myocytes were further cultured for 6 h in serum-free DMEM to eliminate any influence of some factors in the serum.

Confocal Ca²⁺ imaging The cultured NRVM were loaded with 4 µmol/L Fluo-4/AM (Molecular Probes, Eugene, OR, USA) at 37 °C for 30 min, and were then washed with 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid N-(2-Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES)- buffered salt solution (in mmol/L: NaCl 135, KCl 5, MgCl₂ 1, CaCl₂ 1.8, HEPES 10, and glucose 11, with pH 7.4 adjusted by NaOH) for 20 min. All the treatments for each dish were finished within 2 h.

Confocal images of fluo-4 fluorescence (excitation at 488 nm and emission detection at >515 nm) were obtained using a Leica SP2 inverted microscope equipped with a 63×, 1.3 numerical aperture, oil immersion objective. Time- lapsed (xy, 1.63 s/frame) or line-scan (xt, 2 ms/line, 0.15 µm/pixel) images were obtained with 1.5-µm axial resolution. Image data analysis used customer-devised routines coded in the Interactive Data Language Research System. All experiments were performed at room temperatures (22–24 °C).

Materials 5-Bromo-2-deoxyuridine, phenylephrine, 5-methylurapidil (5-Mu), chloroethylclonidine (CEC), BMY 7378 {8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro [4,5]decane-7,9-dione dihydrochloride}, propranolol, and prazosin were purchased from Sigma-Aldrich (St Louis, MO, USA). A61603 {N-[5-(4,5-dihydro-1H-imidazol-2-yl)-2-hydroxy-5,6,7,8-tetra hydronaphthalen-1-yl]methanesul-fonamide hydrobromide} was from Tocris (Ellisville, MO, USA).

Statistical analysis The data were analyzed and presented as mean±SEM. When appropriate, statistical comparison was carried out with 2-way paired or unpaired Student’s t-test or χ² test. The accepted level of significance was P<0.05.

Results

In fluo-4-loaded NRVM, due to spontaneous action potentials as the trigger, rhythmic and spontaneous Ca²⁺ oscillations, were observed at a rate of 6.02±0.58 min^-1 (n=12 experiments). Phenylephrine (PE, 10 µmol/L), a non-subtype specific agonist of the α₁-AR^[17,19,22], increases the frequency of the spontaneous Ca²⁺ transients (Figure 1A, upper panel), which was completely blocked with 1 µmol/L prazosin, an α₁-AR antagonist, but not the β-AR antagonist propranolol at 1 µmol/L. This effect of PE is dose-dependent with an EC₅₀ value (the concentration for inducing 50% of maximal response) of 2.3 µmol/L (Figure 1B). To determine the role of the α₁-AR subtypes in [Ca²⁺]_i regulation, we then examined the effects of subtype-specific antagonists on PE-mediated Ca²⁺ signal. As shown in Figure 1 (1A, bottom panel, 1C), pretreatment of myocytes with 5-Mu for 10 min, a specific inhibitor of the α_1A-AR^[23], caused dose-dependent suppression of the stimulatory response to PE (10 µmol/L) with an IC₅₀ value (the concentration for 50% inhibition of agonist-induced response) of 6.7 nmol/L, and a complete abolishment was seen at a concentration of 30 nmol/L. In contrast, pretreatment of the cells for 30 min with CEC to inhibit the α_1B-AR showed no influence, except that CEC at higher concentration (30 µmol/L) induced a 33.5% inhibition of PE-enhanced Ca²⁺ transients (Figure 1D). The alkylating agent CEC primarily inactivates the α_1B-AR, but studies have shown that this compound can also produce partial inactivation of the other subtypes, especially α_1A-AR, with prolonged exposure at high concentrations^[24,25]. Thus, the partial blockade of the PE effect by CEC at higher concentration is most likely due to its non-specific inhibition of other subtypes. Nevertheless, blockade of the α_1D-AR with BMY 7378 (0.1 µmol/L)^[26,27] demonstrated no any influence in the PE effect (Figure 1D). Presently, the α_1D-AR expressing much less than other subtypes in cardiomyocytes is functionally unknown in the heart^[3,15]. These findings provide clues that the α_1A-AR, not the α_1B-AR and α_1D-AR, may be the primary mediator of PE-regulated spontaneous Ca²⁺ transients.

Figure 1 Effects of different α₁-AR subtype blockade on PE-induced potentiation of spontaneous Ca²⁺ transients in neonatal rat ventricular myocytes. (A) in myocytes loaded with the Ca²⁺ indicator fluo-4, increased spontaneous Ca²⁺ oscillations were observed 3 min after 10 µmol/L PE treatment, and were abolished by the pretreatment of cells with 30 nmol/L 5-Mu for 10 min. (B) dose-dependence of PE effect on spontaneous Ca²⁺ oscillation frequency (EC₅₀=2.3 µmol/L). Data were expressed as the percentage of the control. n=5–9 separated experiments for each PE concentration. (C) concentration-dependent inhibition of 5-Mu on PE induced enhancement of spontaneous Ca²⁺ transients (IC₅₀=6.7 nmol/L). n=6–7 separated experiments for each point. (D) effects of CEC, 5-Mu, BMY 7378, and 0.1 µmol/L BMY 7378+30 nmol/L 5-Mu (pretreated cells for 30, 10, 10 and 10 min, respectively) on PE-potentiated spontaneous Ca²⁺ transients. Data are presented as the percentage of the vehicle control (con, H₂O). ^cP<0.01 vs con; ^fP<0.01 vs PE group. n=6–8 experiments.

At present, the determination of the role of the α_1A-AR versus the α_1B-AR subtypes in mediating physiological responses to α₁-adrenergic stimulation is difficult because of the paucity of highly selective antagonists specific for one subtype over the other. Therefore, to further discriminate the α_1A-AR from other subtypes, we investigated subtype specific agonists in this protocol. So far no specific compound for the α_1B-AR or the α_1D-AR subtype is available, so we examined the effects of A61603, the recently described potent α_1A-adrenergic agonist^[28], and compared the dose-response characteristics of PE and A61603. As shown in Figure 2 (2A,2C), A61603 induced a dose-response increase in spontaneous Ca²⁺ transients with an EC₅₀ value of 6.9 nmol/L, indicating a 330-fold greater potency for PE (2.3 µmol/L). Furthermore, we tried to find the concentration of A61603 at which stimulated Ca²⁺ transients with a similar potency to that of PE so as to evaluate and compare the inhibitory effect of 5-Mu. We observed that 30 nmol/L A61603 potentiated the rate of Ca²⁺ transients with an almost same potency as that of 10 µmol/L PE (2.11 and 2.09 times that of the control, respectively; Figure 2C,2D). Similar as its effect on PE (Figure 1D), 5-Mu (30 nmol/L), pretreatment of cells for 10 min, abolished the increment in Ca²⁺ transients induced by 30 nmol/L A61603 (Figure 2B,2D), while no effect was observed in 10 µmol/L CEC- or 0.1 µmol/L BMY 7378-treated cells (data not shown).

Figure 2 Effect of A61603 on spontaneous Ca²⁺ oscillations in neonatal rat ventricular myocytes. Increased spontaneous Ca²⁺ oscillations were observed 3 min after 30 nmol/L A61603 treatment (A), and were abolished by 30 nmol/L 5-Mu pretreatment of myocytes for 10 min (B). (C) dose-response effect of A61603 on spontaneous Ca²⁺ transients (EC₅₀=6.9 nmol/L). n=7–8 separated experiments for each point. (D) comparable effects of PE (10 µmol/L) and A61603 (30 nmol/L) on spontaneous Ca²⁺ transients and abolishment of these effects by 30 nmol/L 5-Mu, pretreatment of cells for 10 min. Data are presented as the percentage of the vehicle control (con, H₂O). ^cP<0.01 vs con; ^fP<0.01 vs A61603 group. n=6–7 experiments.

As spatial temporal Ca²⁺ sparks or waves constitute the elementary events of Ca²⁺ signaling in response to α₁-adrenergic stimulation inside the cells, we then investigated the characteristics of Ca²⁺ sparks mediated by PE and A61603 in NRVM. With the aid of the line-scan confocal imaging of NRVM (Figure 3A, inset), we found that Ca²⁺ sparks occurred at a frequency of 1.56±0.2/100 µm·s or 1.27±0.15/100 µm·s in the control condition (in the absence of PE or A61603, respectively). PE (10 µmol/L) and A61603 (30 nmol/L) elicited a spark increase by 2.0- and 2.3-fold, respectively (Figure 3A, 3B), while the amplitude, width, and duration of the Ca²⁺ sparks were not altered by either treatment of the agonists. Consistent with the data in global Ca²⁺ transients, the local Ca²⁺ release responses to PE and A61603 could be abolished by 30 nmol/L 5-Mu (Figure 3B, 3C), but not by 10 µmol/L CEC or 0.1 µmol/L BMY 7378 treatment (data not shown). Therefore, the similar responses to A61603 and PE from local Ca²⁺ release to global Ca²⁺ transients, and a complete abolishment of both effects by specific α_1A-AR antagonist, strongly suggest that the α_1A-AR subtype plays a major role in the α₁-AR-associated regulation of intracellular Ca²⁺ signaling in NRVM.

Figure 3 Modulation of local Ca²⁺ sparks by PE and A61603 in neonatal rat ventricular myocytes. (A) typical line-scan images of Ca²⁺ sparks under control conditions (con) and in the presence of PE (10 µmol/L, 3 min, upper panel) or A61603 (30 nmol/L, 3 min, bottom panel). Inset: dashed line shows the position of confocal line-scanning across the nucleus and the cytosol where the images were taken. (B) statistics of spark numbers (events/100 µm·s) from line-scan images in A for the control, PE or A61603, and 5-Mu+PE or A61603 groups as indicated. n=24–30 cells. ^cP<0.01 vs control (con). ^fP<0.01 vs PE or A61603 group, respectively. (C) typical line-scan images of Ca²⁺ sparks under control condition (con) in the presence of 5-Mu (10 min) alone, and PE or A61603 after 5-Mu treatment.

Discussion

Increases in intracellular Ca²⁺ signaling have been implicated to be an essential signal transduction event in the regulation of cardiac functions by α₁-AR stimulation^{[2,7–11,20,21]}. However, the subtype involved is not clear. The present study demonstrates that the stimulatory responses of spontaneous Ca²⁺ oscillations to α₁-AR activation were greatly sensitive to and selectively abolished by the α_1A-AR antagonist, but not by antagonism of the α_1B-AR or the α_1D-AR subtype (Figure 1). Additionally, A61603, the novel α_1A-AR-selective agonist, exhibited a 330-fold greater potency than PE in stimulating spontaneous Ca²⁺ transient activity (Figure 2). This is in agreement with the findings that A61603 produces 340- and 330-fold greater potency than PE in stimulating sarcolemmal Na-H exchange activity in rat ventricular myocytes and in inducing contraction of the rat vas deferens, respectively. These physiological activities of α₁-AR activation are further confirmed to be mediated by α_1A-AR selectively^[22,28]. Furthermore, Ca²⁺ sparks, an important event of the local Ca²⁺ releasing activity, were also stimulated by PE and A61603 with almost an equal potency with that in stimulating Ca²⁺ transients. This response to PE or A61603 is consistently abolished by the α_1A-AR antagonist, but not by α_1B-AR or α_1D-AR inhibition (Figure 3). Taking these results together, these observations provide supportive evidence that α₁-adrenergic stimulation of Ca²⁺ signaling activity is mediated selectively by the α_1A-AR subtype.

Our previous study and those of others have shown that stimulated intracellular Ca²⁺ signaling plays an important role in the induction and perpetuation of cardiac hypertrophy by α₁-AR activation^{[11,12,20,21]}. Importantly, studies have shown that the α_1A-AR subtype is sufficient in inducing hypertrophy in cultured cardiac myocytes^[17,18] and is important in the development of the heart^[6]. Thus, combined with the previous reports, the present study suggests that α_1A-AR-mediated Ca²⁺ signaling response may assume greater significance in hypertrophy formation. Additionally, Ca²⁺ signal abnormity has also been suggested to play a key role in triggering ectopic automaticity in physiological as well as pathological circumstances, such as cardiac ischemia and heart failure^[7,9,10] and α₁-AR activation appears to be one of the important underlying mechanisms in this regard^[7,29–31]. Interestingly, the α_1A-AR has been suggested to be the crucial arrhythmogenic subtype by both in vivo and in vitro studies^[7,9]. Therefore, taken these results together, the finding that the α_1A-AR is the major subtype in intracellular Ca²⁺ signaling regulation during α₁-AR activation may provide significant information for the functional roles of the α₁-AR subtype and an alternate insight into the potential therapeutic candidates in heart remodeling and arrhythmias, particularly in humans as the α_1A-AR appears to be the predominant subtype expressed in the human ventricular myocardium^[32].

In summary, the present study has shown that the α_1A-AR is the predominant subtype in regulating intracellular Ca²⁺ signaling for the α₁-AR activation of neonatal rat ventricular myocytes, which may provide potential information for more specific drug development to hinder the cardiac remodeling process.

Acknowledgement

We thank Dr Qi-hua HE for outstanding technical assistance.

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