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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, α1A-AR,
α1B-AR, and α1D-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 α1A- AR and α1B-AR appear to play major roles in the
heart[3,6,15,16]. More recently however, the
α1A-AR has been demonstrated to sufficiently induce cardiac arrhythmias and
hypertrophy, while the α1B-AR seems less
important[7,17,18]. Furthermore, the avtivation
of α1B-AR even inhibits α1A-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 c2 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
b-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
α1A-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
α1B-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
α1B-AR, but studies have shown that this compound can also produce
partial inactivation of the other subtypes, especially
α1A-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 α1D-AR with BMY 7378 (0.1
µmol/L)[26,27] demonstrated no any influence in the PE effect
(Figure 1D). Presently, the α1D-AR expressing much less than
other subtypes in cardiomyocytes is functionally unknown in
the heart[3,15]. These findings provide clues that the
α1A-AR, not the α1B-AR and
α1D-AR, may be the primary mediator of PE-regulated spontaneous
Ca2+ transients.
At present, the determination of the role of the
α1A-AR versus the α1B-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 α1A-AR from other subtypes, we investigated subtype
specific agonists in this protocol. So far no specific
compound for the α1B-AR or the
α1D-AR subtype is available, so we examined the effects of A61603, the recently described
potent α1A-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).
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
α1A-AR antagonist, strongly suggest that the
α1A-AR subtype plays a major role in the
α1-AR-associated regulation of intracellular
Ca2+ signaling in NRVM.
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
α1A-AR antagonist, but not by antagonism of the
α1B-AR or the α1D-AR subtype (Figure 1). Additionally, A61603, the novel
α1A-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
α1A-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 α1A-AR antagonist, but not
by α1B-AR or α1D-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
α1A-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 α1A-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
α1A-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
α1A-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 α1A-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 α1A-AR appears to be the predominant
subtype expressed in the human ventricular
myocardium[32].
In summary, the present study has shown that the
α1A-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.
References
1 Piascik MT, Perez DM. α1-Adrenergic receptors: new insights
and directions. J Pharmacol Exp Ther 2001; 298: 403_10.
2 Graham RM, Perez DM, Hwa J, Piascik MT.
α1-Adrenergic receptor subtypes: molecular structure, function, and signaling.
Circ Res 1996; 78: 737_49.
3 Lazou A, Fuller SJ, Bogoyevitch MA, Orfali KA, Sugden PH.
Characterization of stimulation of phosphoinositide hydrolysis
by α1-adrenergic agonists in adult rat hearts. Am J Physiol 1994;
267: H970_8.
4 Brodde OE, Michel MC. Adrenergic and muscarinic receptors in
the human heart. Pharmacol Rev 1999; 51: 651_90.
5 Li K, He H, Li C, Sirois P, Rouleau JL. Myocardial
alpha1-adrenoceptor: inotropic effect and physiologic and pathologic
implications. Life Sci 1997; 60: 1305_18.
6 O'Connell TD, Ishizaka S, Nakamura A, Swigart PM, Rodrigo
MC, Simpson GL, et al. The
α1A/C- and α1B-adrenergic receptors
are required for physiological cardiac hypertrophy in the
double-knockout mouse. J Clin Invest 2003; 111: 1783_91.
7 Harrison SN, Autelitano DJ, Wang BH, Milano C, Du XJ,
Woodcock EA. Reduced reperfusion-induced Ins(1,4,5)P3 generation
and arrhythmias in hearts expressing constitutively active
alpha1B-adrenergic receptors. Circ Res 1998; 83: 1232_40.
8 Hunton DL, Lucchesi PA, Pang Y, Cheng X, Dell'Italia LJ,
Marchase RB. Capacitative calcium entry contributes to nuclear
factor of activated T cells nuclear translocation and
hypertrophy in cardiomyocytes. J Biol Chem 2002; 277: 14266_73.
9 Tolg R, Kurz T, Ungerer M, Schreieck J, Gorge B, Richardt G.
Influence of alpha- and beta-adrenoceptor antagonists on
ventricular fibrillation in ischemic rat hearts. Naunyn Schmiedebergs
Arch Pharmacol 1997; 356: 62_8.
10 Mackenzie L, Bootman MD, Laine M, Berridge MJ, Thuring J,
Holmes A, et al. The role of inositol 1,4,5-trisphosphate
receptors in Ca2+ signalling and the generation of arrhythmias in rat
atrial myocytes. J Physiol 2002; 541: 395_409.
11 Liu SJ, Kennedy RH. α1-Adrenergic activation of L-type calcium
current in rat ventricular myocytes: perforated patch clamp
recordings. Am J Physiol 1998; 274: H2203_7.
12 Sugden PH. Signaling in myocardial hypertrophy: life after
calcineurin? Circ Res 1999; 84: 633_46.
13 Frey N, McKinsey TA, Olson EN. Decoding calcium signals
involved in cardiac growth and function. Nat Med 2000; 6:
1221_7.
14 Luther HP, Podlowski S, Schulze W, Morwinski R, Buchwalow I,
Baumann G, et al. Expression of alpha1-adrenergic receptor
subtypes in heart cell culture. Mol Cell Biochem 2001; 224:
69_79.
15 Stewart AF, Rokosh DG, Bailey BA, Karns LR, Chang KC, Long
CS, et al. Cloning of the rat
α1C-adrenergic receptor from cardiac myocytes:
α1C, α1B, and α1D mRNAs are present in cardiac
myocytes but not in cardiac fibroblasts. Circ Res 1994; 75:
796_802.
16 Lemire I, Ducharme A, Tardif JC, Poulin F, Jones LR, Allen BG,
et al. Cardiac-directed overexpression of wild-type
alpha1B-adrenergic receptor induces dilated cardiomyopathy. Am J Physiol
Heart Circ Physiol 2001; 281: H931_8.
17 Autelitano DJ, Woodcock EA. Selective activation of
alpha1A-adrenergic receptors in neonatal cardiac myocytes is sufficient
to cause hypertrophy and differential regulation of
alpha1-adrenergic receptor subtype mRNAs. J Mol Cell Cardiol 1998; 30:
1515_23.
18 Pönicke K, Schlüter KD, Heinroth-Hoffmann I, Seyfarth T,
Goldberg M, Osten B, et al. Noradrenaline-induced increase in
protein synthesis in adult rat cardiomyocytes: involvement of
only α1-adrenoceptors. Naunyn-Schmiedebergs Arch Pharmacol
2001; 364: 444_53.
19 Deng XF, Sculptoreanu A, Mulay S, Peri KG, Li JF, Zheng WH,
et al. Crosstalk between alpha-1A and alpha-1B adrenoceptors in
neonatal rat myocardium: implications in cardiac hypertrophy.
J Pharmacol Exp Ther 1998; 286: 489_96.
20 Luo DL, Gao J, Lan XM, Wang G, Wei S, Xiao RP,
et al. Role of inositol 1,4,5-trisphosphate receptors in
α1-adrenergic receptor-induced cardiomyocyte hypertrophy. Acta Pharmacol Sin
2006; 27: 895_900.
21 Garcia KD, Shah T, Garcia J. Immunolocalization of type 2
inositol 1,4,5-trisphosphate receptors in cardiac myocytes from
newborn mice. Am J Physiol Cell Physiol 2004; 287:
C1048_57.
22 Yokoyama H, Yasutake M, Avkiran M.
α1-Adrenergic stimulation of sarcolemmal
Na+-H+ exchanger activity in rat ventricular
myocytes: evidence for selective mediation by the
a1A-adreno-ceptor subtype. Circ Res 1998; 82: 1078_85.
23 Li HT, Long CS, Gray MO, Rokosh DG, Honbo NY, Karliner JS.
Cross talk between angiotensin AT1 and alpha1-adrenergic
receptors: angiotensin II downregulates alpha1a-adrenergic
receptor subtype mRNA and density in neonatal rat cardiac
myocytes. Circ Res 1997; 81: 396_403.
24 Laz TM, Forray C, Smith KE, Bard JA, Vaysse PJ, Branchek TA,
et al. The rat homologue of the bovine
α1C-adrenergic receptor shows the pharmacological properties of the classical
α1A subtype. Mol Pharmacol 1994; 46: 414_22.
25 Perez DM, Piascik MT, Malik N, Gavin R, Graham RM. Cloning,
expression, and tissue distribution of the rat homolog of the
bovine α1C-adrenergic receptor provide evidence for its
classification as the a1A subtype. Mol Pharmacol 1994; 46: 823_31.
26 Deng XF, Chemtob S, Varma DR. Characterization of
α1D-adreno-ceptor subtype in rat myocardium, aorta and other tissues. Br J
Pharmacol 1996; 119: 269_76.
27 Turnbull L, McCloskey DT, O'Connell TD, Simpson PC, Baker
AJ. α1-Adrenergic receptor responses in
α1AB-AR knockout mouse hearts suggest the presence of
α1D-AR. Am J Physiol Heart Circ Physiol 2003; 284: H1104_H9.
28 Knepper SM, Buckner SA, Brune ME, DeBernardis JF, Meyer
MD, Hancock AA. A-61603, a potent α1-adrenergic receptor
agonist, selective for the α1A receptor subtype. J Pharmacol Exp
Ther 1995; 274: 97_103.
29 Kontula K, Laitinen PJ, Lehtonen A, Toivonen L, Viitasalo M,
Swan H. Catecholaminergic polymorphic ventricular tachycardia:
recent mechanistic insights. Cardiovasc Res 2005; 67: 378_87.
30 Lanzafame AA, Turnbull L, Amiramahdi F, Arthur JF, Huynh H,
Woodcock EA. Inositol phospholipids localized to caveolae in
rat heart are regulated by alpha1-adrenergic receptors and by
ischemia-reperfusion. Am J Physiol Heart Circ Physiol 2006;
290: H2059_65.
31 Ravingerova T, Pancza D, Ziegelhoffer A, Styk J.
Preconditioning modulates susceptibility to ischemia-induced arrhythmias in
the rat heart: the role of alpha-adrenergic stimulation and
K(ATP) channels. Physiol Res 2002; 51: 109_19.
32 Price DT, Lefkowitz RJ, Caron MG, Berkowitz D, Schwinn DA.
Localization of mRNA for three distinct
α1-adrenergic receptor subtypes in human tissues: implications for human
α1-adrenergic physiology. Mol Pharmacol 1994; 45: 171_5.
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