Epinephrine-induced Ca2+ influx in vascular endothelial cells is mediated by CNGA2 channels

doi:10.1016/j.yjmcc.2008.06.005

Journal of Molecular and Cellular Cardiology

Volume 45, Issue 3, September 2008, Pages 437-445

https://doi.org/10.1016/j.yjmcc.2008.06.005 Get rights and content

Abstract

Epinephrine, through its action on β-adrenoceptors, may induce endothelium-dependent vascular dilation, and this action is partly mediated by a cytosolic Ca²⁺ ([Ca²⁺]_i) change in endothelial cells. In the present study, we explored the molecular identity of the channels that mediate epinephrine-induced endothelial Ca²⁺ influx and subsequent vascular relaxation. Patch clamp recorded an epinephrine- and cAMP-activated cation current in the primary cultured bovine aortic endothelial cells (BAECs) and H5V endothelial cells. L-cis-diltiazem and LY-83583, two selective inhibitors for cyclic nucleotide-gated channels, diminished this cation current. Furthermore, this cation current was greatly reduced by a CNGA2-specific siRNA in H5V cells. With the use of fluorescent Ca²⁺ dye, it was found that epinephrine and isoprenaline, a β-adrenoceptor agonist, induced endothelial Ca²⁺ influx in the presence of bradykinin. This Ca²⁺ influx was inhibited by L-cis-diltiazem and LY-83583, and by a β₂-adrenoceptor antagonist ICI-118551. CNGA2-specific siRNA also diminished this Ca²⁺ influx in H5V cells. Furthermore, L-cis-diltiazem and LY-83583 inhibited the endothelial Ca²⁺ influx in isolated mouse aortic strips. L-cis-diltiazem also markedly reduced the endothelium-dependent vascular dilation to isoprenaline in isolated mouse aortic segments. In summary, CNG channels, CNGA2 in particular, mediate β-adrenoceptor agonist-induced endothelial Ca²⁺ influx and subsequent vascular dilation.

Introduction

Epinephrine is the primary catecholamine released from the adrenal medulla in response to low blood glucose, exercise and stress. It exerts a profound effect on vascular system. Depending on vascular beds and chemical concentration, epinephrine may either induce vascular dilation or contraction [1], [2]. While the contractile action of epinephrine is mainly mediated through α-adrenoceptors in vascular smooth muscle cells, the relaxant effect of epinephrine is mainly mediated through β-adrenoceptors, which are located in both vascular smooth muscle and endothelial cells [1]. In many vascular beds, the dilation to β-adrenoceptor agonists is, at least partly, endothelium-dependent and can be attributed to an increased production of nitric oxide (NO) in endothelial cells [3], [4], [5], [6], [7].

cAMP is an important second messenger that participates in the endothelium-dependent vascular dilation. Activation of β-adrenoceptors stimulates adenylyl cyclases, causing subsequent production of cAMP [1]. Elevated cAMP then activates endothelial nitric oxide synthase (eNOS) either via a Ca²⁺-independent pathway that involves protein kinase A [8] or via a Ca²⁺-dependent pathway that involves Ca²⁺-calmodulin [9], [10], [11]. In the latter case, β-adrenoceptor agonists act on endothelial cells to elevate cAMP, which either increases cytosolic Ca²⁺ level ([Ca²⁺]_i) by itself [9] or enhances agonist-induced [Ca²⁺]_i rise [10], [11], both of which result in an increased NO biosynthesis [7], [10], [11].

Little is known about the molecular identity of channels that mediate epinephrine-induced Ca²⁺ influx in endothelial cells. Vascular endothelial cells express multiple Ca²⁺-permeable channels, which include transient receptor potential (TRP) and cyclic nucleotide-gated (CNG) channels [12], [13], [14]. CNG channels are activated by cAMP and cGMP [15], the levels of which are elevated when endothelial cells are exposed to β-adrenoceptor agonists [4], [10], [16], [17]. Therefore, CNG channels could be a potential candidate that mediates β-adrenoceptor agonist-induced Ca²⁺ influx in endothelial cells.

In the present study, we used the methods of patch clamp, Ca²⁺-sensitive fluorescent dye and myograph to study the role of CNG channels in vascular endothelial cells. Our data demonstrated that CNG channels, especially CNGA2, mediate the endothelial Ca²⁺ influx in response to epinephrine and β-adrenoceptor agonists. Furthermore, inhibition of CNG channels greatly reduced isoprenaline-induced vascular dilation in mouse aortic segments.

Section snippets

Cell culture and aortic strip preparation

The animal study was conducted in conformity with the Guide for animal Care and Use of Laboratory Animals published by the US National Institute of Health. The primary cultured BAECs were isolated from bovine aorta as described elsewhere [18]. Briefly, bovine aortic segments were cut open longitudinally. The intima layer was peeled off and then digested with 0.1% collagenase in PBS (in mmol/L: 140 NaCl, 3 KCl, 25 Tris, pH 7.4) for 15 min at 37 °C under vigorous shaking. Dissociated cells were

Role of CNG channels in epinephrine-induced cation current

Whole-cell voltage clamp was used to study the epinephrine-activated cation current in BAECs. The recorded current (Fig. 1A) and the corresponding current–voltage (I–V) relationship (Fig. 1B) showed that the current had slight outward rectification. Epinephrine treatment (1 μmol/L, 5 min) increased the magnitude of the whole-cell current in both inward (negative) and outward (positive) directions (Figs. 1A and B). It is known that epinephrine, via its action on β-adrenergic receptors, causes an

Discussion

The major findings of this study are as follows: Firstly, with the use of whole-cell patch clamp, we recorded a current that was activated by cAMP and epinephrine in the primary cultured BAECs and H5V endothelial cells. We demonstrated that this current was sensitive to selective CNG channel blockers L-cis-diltiazem and LY-83583. Furthermore, a CNGA2-specific siRNA diminished this current in H5V cells. These results strongly suggest that epinephrine activates CNG channels, CNGA2 in particular,

Acknowledgments

We thank the financial support of the Hong Kong Research Grant Council (CUHK4526/06M), Focused Investment Scheme of Chinese University of Hong Kong, and Li Ka Shing Institute of Health Sciences.

References (30)

L. Brawley et al.
Role of endothelium/nitric oxide in atypical beta-adrenoceptor-mediated relaxation in rat isolated aorta
Eur. J. Pharmacol.
(2000)
R. Busse et al.
EDHF: bringing the concepts together
Trends. Pharmacol. Sci.
(2002)
Y. Shimekake et al.
Adrenomedullin stimulates two signal transduction pathways, cAMP accumulation and Ca²⁺ mobilization, in bovine aortic endothelial cells
J. Biol. Chem.
(1995)
S. Guimaraes et al.
Vascular adrenoceptors: an update
Physiol. Rev.
(2001)
A.L. Mark et al.
Differences in direct effects of adrenergic stimuli on coronary, cutaneous, and muscular vessels
J. Clin. Invest.
(1972)
R.M. Priest et al.
Noradrenaline, beta-adrenoceptor mediated vasorelaxation and nitric oxide in large and small pulmonary arteries of the rat
Br. J. Pharmacol.
(1997)
D.W. Gray et al.
Novel signal transduction pathway mediating endothelium-dependent beta-adrenoceptor vasorelaxation in rat thoracic aorta
Br. J. Pharmacol.
(1992)
B. Xu et al.
Nitric oxide-dependent vasodilatation of rabbit femoral artery by beta(2)-adrenergic stimulation or cyclic AMP elevation in vivo
Br. J. Phramacol.
(2000)
A. Ferro et al.
Activation of nitric oxide synthase by beta 2-adrenoceptors in human umbilical vein endothelium in vitro
Br. J. Pharmacol.
(1999)
Y.C. Boo et al.
Shear stress stimulates phosphorylation of eNOS at Ser(635) by a protein kinase A-dependent mechanism
Am. J. Physiol. Heart. Circ. Physiol.
(2002)

T.A. Brock et al.

GTP gamma S loading of endothelial cells stimulates phospholipase C and uncouples ATP receptors

Am. J. Physiol.

(1988)

W.F. Graier et al.

Increases in endothelial cyclic AMP levels amplify agonist-induced formation of endothelium-derived relaxing factor (EDRF)

Biochem. J.

(1992)

K.W. Buchan et al.

Modulation of agonist-induced calcium mobilisation in bovine aortic endothelial cells by phorbol myristate acetate and cyclic AMP but not cyclic GMP

Br. J. Pharmacol.

(1991)

B. Nilius et al.

Ion channels and their functional role in vascular endothelium

Physiol. Rev.

(2001)

K.T. Cheng et al.

Expression of olfactory-type cyclic nucleotide-gated channel (CNGA2) in vascular tissues

Histochem. Cell. Biol.

(2003)

Cited by (30)

Involvement of cyclic nucleotide-gated channels in spontaneous activity generated in isolated interstitial cells of Cajal from the rabbit urethra
2017, European Journal of Pharmacology
Citation Excerpt :
An alternative possibility is that CNG channels could be involved in phenylephrine responses in RUICC. For example, in vascular endothelial cells, responses to epinephrine are inhibited by L-cis-diltiazem and by CNGA2-specific siRNA, demonstrating that CNG channels are actively involved in epinephrine responses in these cells (Shen et al., 2008). Further experiments will be required to investigate these possibilities, but at present an effect of L-cis-diltiazem on the PLC/IP3 pathway cannot be ruled out.
Cyclic nucleotide-gated (CNG) channels are non-selective cation channels that mediate influx of extracellular Na⁺ and Ca²⁺ in various cell types. L-cis-Diltiazem, a CNG channel blocker, inhibits contraction of urethral smooth muscle (USM), however the mechanisms underlying this effect are still unclear. We investigated the possibility that CNG channels contribute to spontaneous pacemaker activity in freshly isolated interstitial cells of Cajal (ICC) isolated from the rabbit urethra (RUICC). Using immunocytochemistry, we found intense CNG1-immunoreactivity in vimentin-immunoreactive RUICC, mainly within patches of the cellular body and processes. In contrast, α-actin immunoreactive smooth muscle cells (SMC) did not show significant reactivity to a specific CNGA1 antibody. Freshly isolated RUICC, voltage clamped at −60 mV, developed spontaneous transient inward currents (STICs) that were inhibited by L-cis-Diltiazem (50 µM). Similarly, L-cis-Diltiazem (50 µM) also inhibited Ca²⁺ waves in isolated RUICC, recorded using a Nipkow spinning disk confocal microscope. L-cis-Diltiazem (50 µM) did not affect caffeine (10 mM)-induced Ca²⁺ transients, but significantly reduced phenylephrine–evoked Ca²⁺ oscillations and inward currents in in RUICC. L-type Ca²⁺ current amplitude in isolated SMC was reduced by ~18% in the presence of L-cis-Diltiazem (50 µM), however D-cis-Diltiazem, a recognised L-type Ca²⁺ channel blocker, abolished L-type Ca²⁺ current but did not affect Ca²⁺ waves or STICs in RUICC. These results indicate that the effects of L-cis-diltiazem on rabbit USM could be mediated by inhibition of CNG1 channels that are present in urethral ICC and therefore CNG channels contribute to spontaneous activity in these cells.
Orai1 forms a signal complex with SK3 channel in gallbladder smooth muscle
2015, Biochemical and Biophysical Research Communications
Citation Excerpt :
Antibody binding was detected by the ECL system. [Ca2+]i was measured as described previously [24]. Briefly, the epithelial layer of the isolated guinea pig gallbladder muscle strips was torn off and the tissue was incubated with 10 μmol/L Fluo-8/AM and 0.02% pluronic F-127 (Invitrogen) for 1 h at 37 °C in Krebs–Henseleit solution.
Orai1 is one of the key components of store-operated Ca²⁺ entry (SOCE) involved in diverse physiological functions. Orai1 may associate with other proteins to form a signaling complex. In the present study, we investigated the interaction between Orai1 and small conductance Ca²⁺-activated potassium channel 3 (SK3). With the use of RNA interference technique, we found that the SOCE and its associated membrane hyperpolarization were reduced while Orai1 was knocked down by a specific Orai1 siRNA in guinea pig gallbladder smooth muscle. However, with the use of isometric tension measurements, our results revealed that agonist-induced muscle contractility was significantly enhanced after Orai1 protein was knocked down or the tissue was treated by SK3 inhibitor apamin, but not affected by larger conductance Ca²⁺-activated potassium channel inhibitor iberiotoxin or intermediate conductance Ca²⁺-activated potassium channel inhibitor TRAM-34. In addition, in the presence of apamin, Orai1 siRNA had no additional effect on agonist-induced contraction. In coimmunoprecipitation experiment, SK3 and Orai1 pulled down each other. These data suggest that, Orai1 physically associated with SK3 to form a signaling complex in gallbladder smooth muscle. Ca²⁺ entry via Orai1 activates SK3, resulting in membrane hyperpolarization in gallbladder smooth muscle. This hyperpolarizing effect of Orai1-SK3 coupling could serve to prevent excessive contraction of gallbladder smooth muscle in response to contractile agonists.
Crystal structure of phospholipase PA2-Vb, a protease-activated receptor agonist from the Trimeresurus stejnegeri snake venom
2014, FEBS Letters
Citation Excerpt :
The pBPB-conjugated PA2-Vb was then applied to the vessel for tension measurements and enzymatic assays. Under a dissecting microscope, the endothelium-denuded aortic strips were placed in an ice-cold PBS solution (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4) and cut into small blocks (2 × 2 mm) [25]. These tissue blocks were then pasted on the bottom of cell culture dishes and cultured in DMEM in a 37 °C incubator.
Phospholipase A₂ (PLA₂) is an important component in snake venoms. Here, an acidic PLA₂, designated PA2-Vb was isolated from the Trimeresurus stejnegeri snake venom. PA2-Vb acts on a protease-activated receptor (PAR-1) to evoke Ca²⁺ release through the inositol 1,4,5-trisphosphate receptor (IP₃R) and induces mouse aorta contraction. PAR-1, phospholipase C and IP₃R inhibitors suppressed PA2-Vb-induced aorta contraction. The crystal structure reveals that PA2-Vb has the typical fold of most snake venom PLA₂. Several PEG molecules bond to a positively charged pocket. The finding offers a novel pharmacological basis of the structure for investigating the PAR-1 receptor and suggests potential applications for PA2-Vb in the vascular system.
AhV_aPA-induced vasoconstriction involves the IP<inf>3</inf>Rs-mediated Ca<sup>2+</sup> releasing
2013, Toxicon
Citation Excerpt :
Only the cells from the first three passages were used for experiments. [Ca2+]i was measured as described elsewhere (Shen et al., 2008). Before experiments, the primary cultured VSMCs were loaded with 10 μM Fluo-8/AM and 0.02% F-127 for 60 min in dark at 37 °C in a NPSS solution (140 mM NaCl, 5 mM KCl, 1 mM CaCl2, 10 mM glucose and 5 mM HEPES, pH 7.4 with NaOH).
AhV_aPA, the acidic PLA₂ purified from Agkistrodon halys pallas venom, was previously reported to possess a strong enzymatic activity and can remarkably induce a further contractile response on the 60 mM K⁺-induced contraction with an EC₅₀ in 369 nM on mouse thoracic aorta rings. In the present study, we found that the p-bromo-phenacyl-bromide (pBPB), which can completely inhibit the enzymatic activity of AhV_aPA, did not significantly reduce the contractile response on vessel rings induced by AhV_aPA, indicating that the vasoconstrictor effects of AhV_aPA are independent of the enzymatic activity. The inhibitor experiments showed that the contractile response induced by AhV_aPA is mainly attributed to the Ca²⁺ releasing from Ca²⁺ store, especially sarcoplasmic reticulum (SR). Detailed studies showed that the Ca²⁺ release from SR is related to the activation of inositol trisphosphate receptors (IP₃Rs) rather than ryanodine receptors (RyRs). Furthermore, the vasoconstrictor effect could be strongly reduced by pre-incubation with heparin, indicating that the basic amino acid residues on the surface of AhV_aPA may be involved in the interaction between AhV_aPA and the molecular receptors. These findings offer new insights into the functions of snake PLA₂ and provide a novel pathogenesis of A. halys pallas venom.
Role of thrombospondin-1 in high-salt–induced mesenteric artery endothelial impairment in rats
2024, Acta Pharmacologica Sinica
Biology of Vascular Smooth Muscle: Vasoconstriction and Dilatation, Second Edition
2022, Biology of Vascular Smooth Muscle: Vasoconstriction and Dilatation, Second Edition

View all citing articles on Scopus

View full text

Original articleEpinephrine-induced Ca2+ influx in vascular endothelial cells is mediated by CNGA2 channels

Abstract

Introduction

Section snippets

Cell culture and aortic strip preparation

Role of CNG channels in epinephrine-induced cation current

Discussion

Acknowledgments

Eur. J. Pharmacol.

Trends. Pharmacol. Sci.

J. Biol. Chem.

Vascular adrenoceptors: an update

Physiol. Rev.

Differences in direct effects of adrenergic stimuli on coronary, cutaneous, and muscular vessels

J. Clin. Invest.

Noradrenaline, beta-adrenoceptor mediated vasorelaxation and nitric oxide in large and small pulmonary arteries of the rat

Br. J. Pharmacol.

Novel signal transduction pathway mediating endothelium-dependent beta-adrenoceptor vasorelaxation in rat thoracic aorta

Br. J. Pharmacol.

Nitric oxide-dependent vasodilatation of rabbit femoral artery by beta(2)-adrenergic stimulation or cyclic AMP elevation in vivo

Br. J. Phramacol.

Activation of nitric oxide synthase by beta 2-adrenoceptors in human umbilical vein endothelium in vitro

Br. J. Pharmacol.

Shear stress stimulates phosphorylation of eNOS at Ser(635) by a protein kinase A-dependent mechanism

Am. J. Physiol. Heart. Circ. Physiol.

GTP gamma S loading of endothelial cells stimulates phospholipase C and uncouples ATP receptors

Am. J. Physiol.

Increases in endothelial cyclic AMP levels amplify agonist-induced formation of endothelium-derived relaxing factor (EDRF)

Biochem. J.

Modulation of agonist-induced calcium mobilisation in bovine aortic endothelial cells by phorbol myristate acetate and cyclic AMP but not cyclic GMP

Br. J. Pharmacol.

Ion channels and their functional role in vascular endothelium

Physiol. Rev.

Expression of olfactory-type cyclic nucleotide-gated channel (CNGA2) in vascular tissues

Histochem. Cell. Biol.

Original article
Epinephrine-induced Ca²⁺ influx in vascular endothelial cells is mediated by CNGA2 channels