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Adrenergic activation of cardiac phospholipase D: role of α1-adrenoceptor subtypes

Kenneth Mier, Dorit Kemken, Hugo A. Katus, Gert Richardt, Thomas Kurz
DOI: http://dx.doi.org/10.1016/S0008-6363(01)00566-1 133-139 First published online: 1 April 2002

Abstract

Objective: Adrenergic stimulation of the heart leads to activation of the phospholipase D signal transduction pathway with formation of the intracellular second messengers phosphatidic acid and diacylglycerol, which may play a role in the development of myocardial hypertrophy by activating mitogen-activated protein kinases and protein kinase C. So far, the adrenergic receptor subtypes mediating activation of cardiac phospholipase D are not known. Methods: We developed an assay for determination of phospholipase D activity in the isolated perfused rat heart. Utilizing the phospholipase D specific transphosphatidylation reaction the stable product phosphatidylethanol (PEtOH) is formed in rat hearts perfused in the presence of 1% ethanol. Myocardial PEtOH formation was used as a marker of phospholipase D activity and was determined by HPLC and evaporative light-scattering detection (PEtOH μg/mg myocardial protein). Results: Basal PEtOH formation in unstimulated hearts was 0.06±0.01 μg/mg. Stimulation of the hearts with norepinephrine resulted in a concentration-dependent phospholipase D activation with a maximum formation of PEtOH (0.17±0.01 μg/mg) at 100 μmol/l norepinephrine. The norepinephrine-induced increase in PLD activity was completely blocked by the α1-adrenoceptor antagonist prazosin and was unaffected by the β-adrenoceptor antagonist propranolol. Further characterisation of α1-adrenoceptor subtypes with selective α1-adrenoceptor antagonists demonstrated a complete inhibition of the norepinephrine-induced phospholipase D activation by WB 4101 (α1A-selective: 0.06±0.01 μg/mg) and by BMY 7378 (α1D-selective: 0.07±0.01 μg/mg). In contrast, the α1B-adrenoceptor antagonist chloroethylclonidine had no inhibitory effect on norepinephrine-stimulated phospholipase D activity (0.14±0.01 μg/mg). Conclusion: Adrenergic activation of the cardiac phospholipase D signal transduction pathway is mediated by α1-adrenoceptors. Here, the α1A-adrenoceptor subtype, but not the α1B-adrenoceptor are coupled to activation of cardiac phospholipase D.

Keywords
  • Adrenergic (ant)agonists
  • Receptors
  • Second messengers
  • Signal transduction

Time for primary review 27 days.

1 Introduction

Phospholipase D (PLD) plays a central role in receptor-mediated breakdown of choline phospholipids [1] implicating the PLD signal transduction pathway as an important regulator of cardiac function [2]. PLD hydrolyses the membrane phosphatidylcholine to generate the second messenger phosphatidic acid. Phosphatidic acid by itself is associated with several cellular modifications, such as stimulation of inositol 1,4,5-trisphosphate production in cardiomyocytes [3], phosphorylation of cardiac proteins [4], increasing intracellular free calcium and contractile force [2], and activation of the mitogen-activated protein kinase cascade [2,5]. In several types of cells and tissues including the heart [6], a major metabolic pathway of phosphatidic acid is rapid dephosphorylation by phosphatidate phosphohydrolase to diacylglycerol, representing an alternative source for diacylglycerol formation to the hydrolysis of phosphatidylinositol bisphosphate by phospholipase C. Thereby, PLD is also linked to myocardial hypertrophy by activating protein kinase C. Diacylglycerol can also intracellularly be converted to phosphatidic acid by diacylglycerol kinase [7]. Thus, formation of phosphatidic acid does not reflect PLD activity due to the intense metabolism of the lipid. PLD, however, has a unique property known as transphosphatidylation, catalyzing the transfer of the aliphatic chain of a primary alcohol to the phosphatidyl moiety of the phosphatidic acid product [1]. The phosphatidylalcohol formed by this transphosphatidylation is less subject to further metabolism and therefore a better index of PLD activity.

Previously, we have shown that norepinephrine increases PLD activity in adult ventricular myocytes via activation of α1-adrenoceptors [8]. We could also demonstrate that myocardial ischemia is associated with an increase in the second messengers phosphatidic acid and diacylglycerol which is due to ischemia-induced release of norepinephrine and subsequent stimulation of myocardial α1-adrenoceptors [9]. So far, it is unknown which α1-adrenoceptor subtype(s) mediate(s) activation of cardiac PLD. Three α1-adrenoceptor subtypes are known and have been pharmacologically defined as α1A, α1B, and α1D [10]. Accordingly, the present study was designed to examine the contribution of adrenoceptor subtypes to stimulation of cardiac PLD activity. In contrast to most studies to date, which have relied on the use of radiolabelling procedures to quantify the lipid mediators, we determined the mass of myocardial phosphatidylethanol formation in rat hearts perfused in the presence of ethanol by a novel HPLC-evaporative light-scattering detection method. Utilizing this assay for determination of PLD activity in the heart, we compared different adrenoceptor antagonists to conclude which adrenoceptor mediates the adrenergic stimulation of cardiac PLD.

2 Methods

2.1 Materials

(−)-Norepinephrine, phosphatidylethanol, dl-propranolol (1-[isopropylamino]-3-[1-naphtyloxy]-2-propanol) and chloroethylclonidine were obtained from Sigma (Taufkirchen, Germany). WB 4101 hydrochloride (2-[2,6-dimethoxyphenoxyethyl]aminomethyl-1,4-benzodioxane), BMY 7378 dihydro-chloride (8-[2-[4-(methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro [4,5]decane-7,9-dione) and prazosin hydrochloride (1-[4-amino-6,7-dimethoxy-2-quinazolinyl]-4-[2-furanylcarbonyl]piperazine) were obtained from Tocris (Bristol, UK). LiChrolut glass columns and LiChroprep Si 60 were obtained from Merck (Darmstadt, Germany). All other chemicals were of analytical grade.

2.2 Isolated perfused heart

Male Wistar rats (180–200 g; Charles River, Germany) were anesthetized by intraperitoneal injection of sodium thiopental (100 mg/kg). After opening the chest, hearts were rapidly excised and placed in cold perfusion buffer. Thereafter, hearts were cannulated via the aorta and retrogradely perfused with modified Krebs-Henseleit solution (composition in mmol/l: Na+ 144, K+ 4.0, Ca2+ 1.85, Mg2+ 1.0, Cl 135, H2PO4 0.22, HCO3 16.7, glucose 11, and EDTA 0.027) at a constant flow of 8 ml/min without external cardiac work. The use of a multichannel peristaltic pump allowed simultaneous perfusion of four hearts. The perfusate was gassed with 95% O2 and 5% CO2. The gas flow was adjusted to achieve a pH of 7.4 in the buffer and the temperature of the perfusate was maintained at 37 °C. During the experiments the hearts were placed within a chamber which was also kept at a temperature of 37 °C. Briefly, all hearts were allowed to stabilize over 15 min before they were subjected to intervention. After the equilibration period 1% ethanol and adrenoceptor antagonists were added to the perfusion buffer and the hearts were perfused for a further 5 min. Finally, norepinephrine was added to the perfusion buffer and the hearts were perfused with all drugs for a further 20 min. After perfusion hearts were frozen in liquid nitrogen.

Animals used in this study were maintained in accordance with the guidelines of the Committee on Animals of the Medizinische Universität zu Lübeck and the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH publication No. 85-23; revised 1996).

2.3 Lipid extraction and partial purification

Frozen hearts were powdered and homogenized in ice-cold buffer (0.1 mmol/l HCl, 0.1 mmol/l Titriplex III). Protein concentration was determined according to Lowry et al. [11]. Total lipids were extracted by slightly modifing the method of Bligh and Dyer [12] followed by solid phase extraction to wash out the majority of neutral lipids. LiChrolut glass columns (12×80 mm) were filled with 0.5 g LiChroprep Si 60. Columns were washed with chloroform and methanol prior to conditioning with chloroform, three bed volumes each. The lipid phase of the Bligh and Dyer extraction was transferred onto the columns. After washing the columns with 3 ml chloroform to elute neutral lipids, polar lipids including PEtOH were eluted with 3 ml methanol. Polar lipid fraction was evaporated to dryness under N2 stream, and the residue dissolved in 100 μl hexane–isopropanol–acetic acid (50:50:1, v/v/v) plus triethylamine (0.05%, v/v) prior to chromatography.

2.4 HPLC separation and quantification

Lipid separation was accomplished by normal-phase HPLC on a LiChrosphere 100 Diol (5 μm) 250×4 mm column (Merck) according to a slightly modified method of Silversand and Haux [13]. The analyses were carried out by binary gradient elution with mobile phase solvents of (A) hexane–isopropanol–acetic acid (82:17:1, v/v/v) and (B) isopropanol–water–acetic acid (85:14:1, v/v/v) at a flow rate of 1 ml/min. Triethylamine (0.08%, v/v) was added to the solvents. The gradient profile started at 5% for solvent B and was increased to 40% B in 25 min, after which it was increased to 100% B in 5 min. Aliquots (50 μl) of lipid samples were injected onto the column, which was kept at a temperature of 45 °C for all runs. The HPLC system was interfaced with an evaporative light-scattering detector (Sedex 45; S.E.D.E.R.E, Vitry-sur-Seine, France). The detector signal was recorded and integrated by a personal computer and a chromatography data software program (Andromeda, Techlab, Germany). The detection limit of the assay is ∼200 ng PEtOH in the HPLC system.

2.5 Statistical analysis

Data are presented as mean±S.D. Statistical calculation was done by one-way analysis of variance (ANOVA) and use of Bonferroni's multiple test for post hoc analysis. A P-value <0.05 was considered statistically significant. The half maximum effective concentration (EC50) was determined with a sigmoid curve fitting routine in the computer program.

3 Results

3.1 Quantification of PEtOH formation by evaporative light-scattering HPLC

Fig. 1 shows the chromatographic separation of lipids extracted from rat hearts perfused in the presence of 1% ethanol. Compared to the unstimulated heart (control) there was a marked increase in the chromatographic peak at ∼7 min in the heart stimulated with norepinephrine (10 μmol/l). This peak could be identified as PEtOH by addition of 1 μg PEtOH standard to the lipids extracted from a stimulated heart prior to chromatography.

Fig. 1

Resolution of phosphatidylethanol (PEtOH) from other lipids by high-performance liquid chromatography (HPLC) and quantification by evaporative light-scattering detection. Total myocardial lipids were purified by solid-phase extraction and separated by HPLC as described in Methods. (A) Representative chromatograms (overlay of three chromatograms) of lipids extracted from rat hearts perfused in the presence of 1% ethanol. Hearts were perfused for 20 min without (control) and with (stimulated) norepinephrine (10 μmol/l) in the perfusion buffer. For peak identification PEtOH standard (1 μg) was added to the sample prior to chromatography (stimulated+1 μg PEtOH). (B) The standard curve of PEtOH quantified by evaporative light-scattering detection. Each data point is representative of at least three similar experiments.

The recovery of added PEtOH was examined and verified by adding selected amounts of exogenous PEtOH standard to aliquots of myocardial lipids and the samples were then treated as described in Methods. The recovery of PEtOH was 80±12% based on the mass of exogenous PEtOH added to each sample. When the standard PEtOH was subjected to HPLC and evaporative light-scattering detection, a standard curve exhibiting a correlation coefficient of 0.998 (five standards in a range from 0.25 to 2 μg PEtOH, n=4 per standard concentration) was obtained (Fig. 1).

3.2 Stimulation of cardiac PLD activity by norepinephrine

Basal PEtOH formation in unstimulated hearts was 0.06±0.01 μg/mg myocardial protein. Stimulation of the hearts with norepinephrine resulted in a concentration-dependent PLD activation with a significant increase of PEtOH formation at 1 μmol/l norepinephrine and a maximum effect at a concentration of 100 μmol/l, the highest concentration of norepinephrine tested (Fig. 2). The concentration required for half maximum increase in PLD activity (EC50) was calculated to be 0.4 μmol/l of norepinephrine.

Fig. 2

Effect of selected norepinephrine concentrations on cardiac PLD activity. Rat hearts were perfused in the presence of 1% ethanol for 20 min without (control) and with the indicated concentrations of norepinephrine in the perfusion buffer. Total lipids were extracted and the mass of PEtOH was determined using HPLC coupled to evaporative light-scattering detection. Each bar represents data of four experiments. Mean±S.D. *P<0.05.

3.3 Influence of adrenergic receptor blocking agents

When rat hearts were perfused in the presence of 1% ethanol without (control) or with norepinephrine (10 μmol/l) for 20 min, PEtOH formation increased from 0.06±0.02 μg/mg (control) to 0.14±0.02 μg/mg (norepinephrine) (Fig. 3). No inhibition was observed in the presence of the β-adrenoceptor antagonist propranolol (10 μmol/l). In contrast, the increase in PEtOH in response to norepinephrine was completely abolished by the α1-adrenoceptor antagonist prazosin (10 μmol/l). Thus, adrenergic activation of cardiac PLD is mediated through the α1-adrenoceptor.

Fig. 3

Influence of α1-adrenoceptor blockade and β-adrenoceptor blockade on cardiac PLD activity during norepinephrine stimulation of rat hearts. Hearts were perfused in the presence of 1% ethanol without (control) and with 10 μmol/l norepinephrine for 20 min, either alone or together with the β-adrenoceptor antagonist propranolol (10 μmol/l) or with the α1-adrenoceptor antagonist prazosin (10 μmol/l) in the perfusion buffer. Total lipids were extracted and the mass of PEtOH was determined using HPLC coupled to evaporative light-scattering detection. Each bar represents data of four experiments. Mean±S.D. *P<0.05.

For further characterisation of α1-adrenoceptor subtypes mediating activation of cardiac PLD we used subtype-selective α1-adrenoceptor antagonists (Fig. 4). The α1A-adrenoceptor subtype selective antagonist WB 4101 decreased the norepinephrine-induced increase in PEtOH formation to basal levels. Likewise, when rat hearts were perfused with the α1D-adrenoceptor antagonist BMY 7378 the norepinephrine-induced PLD activation was completely inhibited. In contrast, the α1B-adrenoceptor antagonist chloroethylclonidine had no inhibitory effect on the increase in PEtOH formation in response to norepinephrine.

Fig. 4

Influence of α1-adrenoceptor subtype blockade on cardiac PLD activity during norepinephrine stimulation of rat hearts. Hearts were perfused in the presence of 1% ethanol without (control) and with 10 μmol/l norepinephrine for 20 min. Selective α1-adrenoceptor antagonists (α1A: WB 4101; α1B: chloroethylclonidine (CEC); α1D: BMY 7378) at a concentration of 10 μmol/l were added to the perfusion buffer 5 min prior to norepinephrine stimulation. Total lipids were extracted and the mass of PEtOH was determined using HPLC coupled to evaporative light-scattering detection. Each bar represents data of four experiments. Mean±S.D. *P<0.05.

4 Discussion

This paper is the first describing the contribution of α1-adrenoceptor subtypes to the adrenergic activation of cardiac phospholipase D (PLD) in the heart. The present study demonstrates that adrenergic activation of the cardiac PLD signal transduction pathway is mediated by α1-adrenoceptors but not the β-adrenoceptor. Further characterization of α1-adrenoceptor subtypes revealed that the α1A-adrenoceptor subtype, but not the α1B-adrenoceptor, are coupled to activation of cardiac PLD.

The present findings were obtained using measurement of the mass of phosphatidylethanol rather than radiolabelling procedures. Utilizing the PLD-specific transphosphatidylation reaction we developed a novel assay for determination of PLD activity in vivo. The stable product phosphatidylethanol (PEtOH) was formed in rat hearts perfused in the presence of 1% ethanol. Myocardial PEtOH formation was used as a marker of PLD activity and was quantitated by HPLC and evaporative light-scattering detection. Most studies to date have relied on the use of radiolabels to tag, follow, and quantify the lipid mediators. However, most cellular radiolabelling procedures do not permit an easily obtainable estimation of the mass level of the second messenger because equilibrium labelling is often not achieved, particularly in perfused organs. Furthermore, unlabelled pools involved in the signalling pathway would not be detected. The assay described here for the quantification of phosphatidylethanol offers improvements over most assays used previously. The primary advantage is the direct measurement of the mass of the lipid.

PEtOH was exclusively formed in hearts in the presence of ethanol in the perfusion buffer. However, even in the absence of adrenergic stimulation we found myocardial PEtOH formation, indicating basal PLD activity in the isolated heart. As shown previously in adult rabbit ventricular myocytes [8], norepinephrine concentration-dependently stimulated PLD activity in perfused rat hearts, and this adrenergic activation of PLD was completely blocked by α1-adrenoceptor antagonism.

In the adult rat heart α1A- and α1B-adrenoceptors account for most α1-adrenoceptors, whereas the expression of α1D-adrenoceptor protein is minimal despite the expression of its mRNA [10,14]. Studies with expressed cloned α1-adrenoceptor subtypes demonstrate that all three subtypes can couple not only to phospholipase C activation but also to activation of PLD [15–17]. The rank order of the coupling efficiency of α1-adrenoceptor subtypes for activating PLD in transfected cell lines is α1A1B1D, although this may not apply to cells expressing native α1-adrenoceptors [16]. Thus, in the present study utilizing α1-adrenoceptor subtype selective antagonists, we have found coupling of the α1A-, and α1D-, but not the α1B-adrenoceptor to cardiac PLD. There is, however, some limitation due to the fact that we did not examine concentration–response relationships of each α1-adrenoceptor antagonist. Although BMY 7378 has been shown to be a highly selective antagonist at the α1D-adrenoceptor with ∼100-fold higher affinity for the α1D than the α1A or α1B subtype [18], we can not completely exclude additional blockade of the α1A-adrenoceptor subtype at the concentration of BMY 7378 used in the present study. Likewise, our finding that either the alpha1A-adrenoceptor antagonist WB 4101 or BMY 7378 inhibited norepinephrine-stimulated PLD activity completely may be explained by a non-selective effect of BMY 7378 for the alpha1D-adrenoceptor. The finding that chloroethylclonidine, a selective α1B-adrenoceptor antagonist in intact cells [19], had no effect on norepinephrine-induced PLD activity strongly implies that cardiac α1B-adrenoceptors are not coupled to PLD in the rat heart.

Several lines of evidence indicate that distinct α1-adrenoceptor subtypes play different roles in cardiac function. Both α1A- and α1B-adrenoceptors mediate positive inotropic responses [10,20,21], but α1B-adrenoceptors seem more important [22,23]. While a role for α1A-adrenoceptors in precipitating ischemic arrhythmias has been demonstrated, the α1B-adrenoceptor might have a protective effect on ischemia-induced arrhythmias [24–26]. Both the α1A- and α1B-adrenoceptor have also been reported to induce myocyte growth and hypertrophy [27–29], although recent findings in transgenic mice with cardiac-targeted overexpression of the α1A-adrenoceptor failed to demonstrate involvement of this subtype in the development of myocardial hypertrophy [21]. PLD signal transduction has been implicated as an important regulator of cardiac function in myocardial ischemia [30], ischemic preconditioning [31,32], and in the pathophysiology of myocardial hypertrophy [33,34]. Thus, the finding in the present study of differential coupling of α1-adrenoceptor subtypes to PLD signal transduction may contribute to a better understanding of the different roles distinct α1-adrenoceptors play in cardiac function.

Acknowledgments

This study was supported by a grant from the Deutsche Forschungsgemeinschaft (KU 774/2-1). We are very grateful to Cindy Krause, Ines Stölting, and Isabel Weber for their technical assistance.

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View Abstract